Andrew McDavid authored on 19/06/2019 14:15:03
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# CellaRepertorium |
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-This package contains methods for clustering and analyzing single cell RepSeq data, especially as generated by [10X genomics VDJ solution](https://support.10xgenomics.com/single-cell-vdj). |
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+This package contains methods for clustering and analyzing single cell RepSeq data, especially as generated by [10X genomics VDJ solution](https://support.10xgenomics.com/single-cell-vdj). |
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+ |
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+## Installation |
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+ |
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+``` |
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+devtools::install_github('amcdavid/CellaRepertorium')
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+``` |
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+ |
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+Requires R>=3.5. |
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+ |
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+## Data requirements and package structure |
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+ |
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+The fundamental unit is the **contig**, which is a section of contiguously stitched reads from a single **cell**. Each contig belongs to one (and only one) cell, however, cells generate multiple contigs. Contigs can also belong to a **cluster**. Because of these two many-to-one mappings, these data can be thought as a series of ragged arrays. The links between them mean they are relational data. |
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+ |
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+[A schematic of contigs and cells should go here] |
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+ |
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+A `ContigCellDB` object wraps each of these objects as a sequence of three `data.frame`s (well, `tibble`s, actually). `ContigCellDB` also tracks columns (keys) that unique identify each row in each of these tables. The `contig_tbl` is the `tibble` containing **contigs**, the `cell_tbl` contains the **cells**, and the `cluster_tbl` contains the **clusters**. The `contig_pk`, `cell_pk` and `cluster_pk` identify the columns that identify a contig, cell and cluster, respectively, and must be unique in each of the respective tables. |
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+The tables are kept in sync so that subsetting the contigs will subset the cells, and clusters, and vice-versa. |
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+ |
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+[A schematic showing table relations should go here] |
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+ |
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+Of course, each of these tables can contain many other columns that will serve as covariates for various analysis, such as the CDR3 sequence, or the identity of the V, D and J regions. Various derived quantities that describe cells and clusters can also be calculated, and added to these tables, such as the medoid of a cluster. |
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## Functions |
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+[a screencap of something interesting?] |
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+ |
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* `cdhit`: An R interface to CDhit, which was originally ported by Thomas Lin Pedersen. |
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* `fine_cluster`: clustering CDR3 by edit distances (possibly using empirical amino acid substitution matrices) |
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* `cluster_permute_test`: permutation tests of cluster statistics |
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+ |
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+ <span class="version label label-default" data-toggle="tooltip" data-placement="bottom" title="Released version">0.3.1</span> |
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+ <li> |
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+ <a href="../articles/mouse_tcell_qc.html">Quality control and Exploration of UMI-based repertoire data</a> |
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+ <div class="col-md-9 contents"> |
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+ <div class="page-header toc-ignore"> |
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+ <h1>Clustering repertoire via CDR3 sequences</h1> |
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+ |
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+ |
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+ <small class="dont-index">Source: <a href="https://github.com/amcdavid/CellaRepertorium/blob/master/vignettes/cdr3_clustering.Rmd"><code>vignettes/cdr3_clustering.Rmd</code></a></small> |
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+ <div class="hidden name"><code>cdr3_clustering.Rmd</code></div> |
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+ |
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+ </div> |
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+ |
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+ |
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+ |
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+<div class="sourceCode" id="cb1"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb1-1" data-line-number="1"><span class="co">#load_all()</span></a> |
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+<a class="sourceLine" id="cb1-2" data-line-number="2"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(CellaRepertorium)</a> |
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+<a class="sourceLine" id="cb1-3" data-line-number="3"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(dplyr)</a> |
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+<a class="sourceLine" id="cb1-4" data-line-number="4"><span class="co">#> </span></a> |
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+<a class="sourceLine" id="cb1-5" data-line-number="5"><span class="co">#> Attaching package: 'dplyr'</span></a> |
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+<a class="sourceLine" id="cb1-6" data-line-number="6"><span class="co">#> The following objects are masked from 'package:stats':</span></a> |
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+<a class="sourceLine" id="cb1-7" data-line-number="7"><span class="co">#> </span></a> |
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+<a class="sourceLine" id="cb1-8" data-line-number="8"><span class="co">#> filter, lag</span></a> |
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+<a class="sourceLine" id="cb1-9" data-line-number="9"><span class="co">#> The following objects are masked from 'package:base':</span></a> |
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+<a class="sourceLine" id="cb1-10" data-line-number="10"><span class="co">#> </span></a> |
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+<a class="sourceLine" id="cb1-11" data-line-number="11"><span class="co">#> intersect, setdiff, setequal, union</span></a> |
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+<a class="sourceLine" id="cb1-12" data-line-number="12"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(ggplot2)</a> |
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+<a class="sourceLine" id="cb1-13" data-line-number="13"><span class="co">#> Registered S3 methods overwritten by 'ggplot2':</span></a> |
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+<a class="sourceLine" id="cb1-14" data-line-number="14"><span class="co">#> method from </span></a> |
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+<a class="sourceLine" id="cb1-15" data-line-number="15"><span class="co">#> [.quosures rlang</span></a> |
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+<a class="sourceLine" id="cb1-16" data-line-number="16"><span class="co">#> c.quosures rlang</span></a> |
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+<a class="sourceLine" id="cb1-17" data-line-number="17"><span class="co">#> print.quosures rlang</span></a> |
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+<a class="sourceLine" id="cb1-18" data-line-number="18"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(readr)</a> |
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+<a class="sourceLine" id="cb1-19" data-line-number="19"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(tidyr)</a> |
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+<a class="sourceLine" id="cb1-20" data-line-number="20"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(stringr)</a> |
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+<a class="sourceLine" id="cb1-21" data-line-number="21"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(purrr)</a></code></pre></div> |
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+<div id="load-filtered-contig-files" class="section level1"> |
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+<h1 class="hasAnchor"> |
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+<a href="#load-filtered-contig-files" class="anchor"></a>Load filtered contig files</h1> |
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+<div class="sourceCode" id="cb2"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb2-1" data-line-number="1"><span class="kw"><a href="https://www.rdocumentation.org/packages/utils/topics/data">data</a></span>(contigs_qc)</a> |
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+<a class="sourceLine" id="cb2-2" data-line-number="2">MIN_CDR3_AA =<span class="st"> </span><span class="dv">6</span></a> |
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+<a class="sourceLine" id="cb2-3" data-line-number="3"></a> |
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+<a class="sourceLine" id="cb2-4" data-line-number="4"></a> |
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+<a class="sourceLine" id="cb2-5" data-line-number="5">cdb =<span class="st"> </span><span class="kw"><a href="../reference/ContigCellDB-fun.html">ContigCellDB_10XVDJ</a></span>(contigs_qc, <span class="dt">contig_pk =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'barcode'</span>, <span class="st">'pop'</span>, <span class="st">'sample'</span>, <span class="st">'contig_id'</span>), <span class="dt">cell_pk =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'barcode'</span>, <span class="st">'pop'</span>, <span class="st">'sample'</span>))</a> |
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+<a class="sourceLine" id="cb2-6" data-line-number="6"></a> |
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+<a class="sourceLine" id="cb2-7" data-line-number="7">cdb<span class="op">$</span>contig_tbl =<span class="st"> </span>dplyr<span class="op">::</span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(cdb<span class="op">$</span>contig_tbl, full_length, productive <span class="op">==</span><span class="st"> 'True'</span>, high_confidence, chain <span class="op">!=</span><span class="st"> 'Multi'</span>, <span class="kw"><a href="https://stringr.tidyverse.org/reference/str_length.html">str_length</a></span>(cdr3) <span class="op">></span><span class="st"> </span>MIN_CDR3_AA) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>( <span class="dt">fancy_name =</span> <span class="kw"><a href="../reference/fancy_name_contigs.html">fancy_name_contigs</a></span>(., <span class="kw"><a href="https://stringr.tidyverse.org/reference/str_c.html">str_c</a></span>(pop, <span class="st">'_'</span>, sample)))</a></code></pre></div> |
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+<p>good chains (either TRA or TRB); each cell can appear more than once.</p> |
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+</div> |
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+<div id="chain-pairings" class="section level1"> |
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+<h1 class="hasAnchor"> |
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+<a href="#chain-pairings" class="anchor"></a>Chain pairings</h1> |
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+<div class="sourceCode" id="cb3"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb3-1" data-line-number="1">paired_chain =<span class="st"> </span><span class="kw"><a href="../reference/enumerate_pairing.html">enumerate_pairing</a></span>(cdb, <span class="dt">chain_recode_fun =</span> <span class="st">'guess'</span>)</a> |
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+<a class="sourceLine" id="cb3-2" data-line-number="2"></a> |
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+<a class="sourceLine" id="cb3-3" data-line-number="3"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(paired_chain, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/interaction">interaction</a></span>(sample, pop), <span class="dt">fill =</span> pairing)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_bar.html">geom_bar</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/facet_wrap.html">facet_wrap</a></span>(<span class="op">~</span>canonical, <span class="dt">scale =</span> <span class="st">'free_x'</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/coord_flip.html">coord_flip</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_minimal</a></span>()</a></code></pre></div> |
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+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-3-1.png" width="700"></p> |
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+</div> |
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+<div id="cluster-cdr3-protein-sequences" class="section level1"> |
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+<h1 class="hasAnchor"> |
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+<a href="#cluster-cdr3-protein-sequences" class="anchor"></a>Cluster CDR3 protein sequences</h1> |
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+<div class="sourceCode" id="cb4"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb4-1" data-line-number="1"></a> |
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+<a class="sourceLine" id="cb4-2" data-line-number="2">aa80 =<span class="st"> </span>CellaRepertorium<span class="op">:::</span><span class="kw"><a href="../reference/cdhit.html">cdhit_ccdb</a></span>(cdb, <span class="st">'cdr3'</span>, <span class="dt">type =</span> <span class="st">'AA'</span>, <span class="dt">cluster_name =</span> <span class="st">'aa80'</span>, <span class="dt">identity =</span> <span class="fl">.8</span>)</a> |
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+<a class="sourceLine" id="cb4-3" data-line-number="3">aa80 =<span class="st"> </span><span class="kw"><a href="../reference/fine_clustering.html">fine_clustering</a></span>(aa80, <span class="dt">sequence_key =</span> <span class="st">'cdr3'</span>, <span class="dt">type =</span> <span class="st">'AA'</span>, <span class="dt">keep_clustering_details =</span> <span class="ot">TRUE</span>)</a> |
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+<a class="sourceLine" id="cb4-4" data-line-number="4"><span class="co">#> Calculating intradistances on 988 clusters.</span></a> |
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+<a class="sourceLine" id="cb4-5" data-line-number="5"><span class="co">#> Summarizing</span></a> |
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+<a class="sourceLine" id="cb4-6" data-line-number="6"></a> |
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+<a class="sourceLine" id="cb4-7" data-line-number="7"></a> |
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+<a class="sourceLine" id="cb4-8" data-line-number="8"><span class="co"># This maybe should be a turned into a function </span></a> |
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+<a class="sourceLine" id="cb4-9" data-line-number="9"><span class="co"># Other plots should be considered:</span></a> |
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+<a class="sourceLine" id="cb4-10" data-line-number="10"><span class="co"># That show how clusters are split between samples, chains, etc</span></a> |
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+<a class="sourceLine" id="cb4-11" data-line-number="11"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(aa80<span class="op">$</span>cluster_tbl <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(n_cluster<span class="op">></span><span class="dv">1</span>) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://tidyr.tidyverse.org/reference/gather.html">gather</a></span>(key, value, <span class="op">-</span>aa80, <span class="op">-</span>fc) , <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> value))<span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/facet_wrap.html">facet_wrap</a></span>(<span class="op">~</span>key, <span class="dt">scales =</span> <span class="st">'free'</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_histogram.html">geom_histogram</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/scale_continuous.html">scale_y_sqrt</a></span>()</a> |
|
| 148 |
+<a class="sourceLine" id="cb4-12" data-line-number="12"><span class="co">#> `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.</span></a></code></pre></div> |
|
| 149 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-4-1.png" width="700"></p> |
|
| 150 |
+<p>We cluster the CDR3 translated amino acid residues with the program <a href="http://weizhongli-lab.org/cdhit_suite/cgi-bin/index.cgi?cmd=cd-hit">CD-HIT</a>. A sequence is included in a cluster if it matches by 100% similiarity and has the same CDR3 length. Note that this can and should be relaxed – especially in the beta chain we see “near clones” that only differ by a residue or two, seemingly in stylized places.</p> |
|
| 151 |
+</div> |
|
| 152 |
+<div id="cluster-cdr3-dna-sequences" class="section level1"> |
|
| 153 |
+<h1 class="hasAnchor"> |
|
| 154 |
+<a href="#cluster-cdr3-dna-sequences" class="anchor"></a>Cluster CDR3 DNA sequences</h1> |
|
| 155 |
+<div class="sourceCode" id="cb5"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb5-1" data-line-number="1">cdb =<span class="st"> </span>CellaRepertorium<span class="op">:::</span><span class="kw"><a href="../reference/cdhit.html">cdhit_ccdb</a></span>(cdb, <span class="st">'cdr3_nt'</span>, <span class="dt">type =</span> <span class="st">'DNA'</span>, <span class="dt">cluster_name =</span> <span class="st">'DNA97'</span>, <span class="dt">identity =</span> <span class="fl">.965</span>, <span class="dt">min_length =</span> MIN_CDR3_AA<span class="op">*</span><span class="dv">3-1</span>, <span class="dt">G =</span> <span class="dv">1</span>)</a> |
|
| 156 |
+<a class="sourceLine" id="cb5-2" data-line-number="2">cdb =<span class="st"> </span><span class="kw"><a href="../reference/fine_clustering.html">fine_clustering</a></span>(cdb, <span class="dt">sequence_key =</span> <span class="st">'cdr3_nt'</span>, <span class="dt">type =</span> <span class="st">'DNA'</span>)</a> |
|
| 157 |
+<a class="sourceLine" id="cb5-3" data-line-number="3"><span class="co">#> Calculating intradistances on 1342 clusters.</span></a> |
|
| 158 |
+<a class="sourceLine" id="cb5-4" data-line-number="4"><span class="co">#> Summarizing</span></a> |
|
| 159 |
+<a class="sourceLine" id="cb5-5" data-line-number="5"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(cdb<span class="op">$</span>cluster_tbl <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(n_cluster<span class="op">></span><span class="dv">1</span>) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://tidyr.tidyverse.org/reference/gather.html">gather</a></span>(key, value, <span class="op">-</span>DNA97) , <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> value))<span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/facet_wrap.html">facet_wrap</a></span>(<span class="op">~</span>key, <span class="dt">scales =</span> <span class="st">'free'</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_histogram.html">geom_histogram</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/scale_continuous.html">scale_y_sqrt</a></span>()</a> |
|
| 160 |
+<a class="sourceLine" id="cb5-6" data-line-number="6"><span class="co">#> `stat_bin()` using `bins = 30`. Pick better value with `binwidth`.</span></a></code></pre></div> |
|
| 161 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-5-1.png" width="700"></p> |
|
| 162 |
+<p>We can also cluster by DNA identity.</p> |
|
| 163 |
+<div class="sourceCode" id="cb6"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb6-1" data-line-number="1">germline_cluster =<span class="st"> </span>CellaRepertorium<span class="op">:::</span><span class="kw">cluster_germline</span>(cdb, <span class="dt">segment_keys =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'v_gene'</span>, <span class="st">'j_gene'</span>, <span class="st">'chain'</span>), <span class="dt">cluster_name =</span> <span class="st">'segment_idx'</span>)</a> |
|
| 164 |
+<a class="sourceLine" id="cb6-2" data-line-number="2"><span class="co">#> Warning in replace_cluster_tbl(ccdb, cluster_tbl, cl_con_tbl, cluster_pk =</span></a> |
|
| 165 |
+<a class="sourceLine" id="cb6-3" data-line-number="3"><span class="co">#> cluster_name): Replacing `cluster_tbl` with key ccdb$cluster_pk</span></a></code></pre></div> |
|
| 166 |
+<p>And by other features of the contigs. Here we cluster each contig based on the chain and V-J genes. This gives us the set of observed V-J pairings:</p> |
|
| 167 |
+<div class="sourceCode" id="cb7"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb7-1" data-line-number="1">germline_cluster =<span class="st"> </span><span class="kw"><a href="../reference/fine_clustering.html">fine_clustering</a></span>(germline_cluster, <span class="dt">sequence_key =</span> <span class="st">'cdr3_nt'</span>, <span class="dt">type =</span> <span class="st">'DNA'</span>)</a> |
|
| 168 |
+<a class="sourceLine" id="cb7-2" data-line-number="2"><span class="co">#> Calculating intradistances on 700 clusters.</span></a> |
|
| 169 |
+<a class="sourceLine" id="cb7-3" data-line-number="3"><span class="co">#> Summarizing</span></a> |
|
| 170 |
+<a class="sourceLine" id="cb7-4" data-line-number="4"><span class="co">#> Warning in left_join_warn(d_medoid, contig_tbl, by = ccdb$contig_pk,</span></a> |
|
| 171 |
+<a class="sourceLine" id="cb7-5" data-line-number="5"><span class="co">#> overwrite = TRUE): Overwriting fields d(medoid), is_medoid in table</span></a> |
|
| 172 |
+<a class="sourceLine" id="cb7-6" data-line-number="6"><span class="co">#> contig_tbl</span></a> |
|
| 173 |
+<a class="sourceLine" id="cb7-7" data-line-number="7"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(germline_cluster<span class="op">$</span>cluster_tbl <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(chain <span class="op">==</span><span class="st"> 'TRB'</span>), <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> v_gene, <span class="dt">y =</span> j_gene, <span class="dt">fill =</span> n_cluster)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_tile.html">geom_tile</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/theme.html">theme</a></span>(<span class="dt">axis.text.x =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/element.html">element_text</a></span>(<span class="dt">angle =</span> <span class="dv">90</span>))</a></code></pre></div> |
|
| 174 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-7-1.png" width="700"></p> |
|
| 175 |
+<p>Number of pairs</p> |
|
| 176 |
+<div class="sourceCode" id="cb8"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb8-1" data-line-number="1"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(germline_cluster<span class="op">$</span>cluster_tbl <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(chain <span class="op">==</span><span class="st"> 'TRB'</span>), <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> v_gene, <span class="dt">y =</span> j_gene, <span class="dt">fill =</span> avg_distance)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_tile.html">geom_tile</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/theme.html">theme</a></span>(<span class="dt">axis.text.x =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/element.html">element_text</a></span>(<span class="dt">angle =</span> <span class="dv">90</span>))</a></code></pre></div> |
|
| 177 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-8-1.png" width="700"></p> |
|
| 178 |
+<p>Average Levenstein distance of CDR3 within each pair</p> |
|
| 179 |
+<div class="sourceCode" id="cb9"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb9-1" data-line-number="1"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(ggdendro)</a> |
|
| 180 |
+<a class="sourceLine" id="cb9-2" data-line-number="2"></a> |
|
| 181 |
+<a class="sourceLine" id="cb9-3" data-line-number="3"><span class="co"># This should be turned into a function in the package somehow</span></a> |
|
| 182 |
+<a class="sourceLine" id="cb9-4" data-line-number="4"><span class="co"># But plot arguments will be super-variable</span></a> |
|
| 183 |
+<a class="sourceLine" id="cb9-5" data-line-number="5"><span class="co"># Maybe just return the `hc` object?</span></a> |
|
| 184 |
+<a class="sourceLine" id="cb9-6" data-line-number="6">dendro_plot =<span class="st"> </span><span class="cf">function</span>(ccdb, idx, <span class="dt">method =</span> <span class="st">'complete'</span>){</a>
|
|
| 185 |
+<a class="sourceLine" id="cb9-7" data-line-number="7"> h =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(ccdb<span class="op">$</span>cluster_tbl, <span class="op">!!</span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/tidyeval.html">sym</a></span>(ccdb<span class="op">$</span>cluster_pk) <span class="op">==</span><span class="st"> </span>idx) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/pull.html">pull</a></span>(fc) <span class="op">%>%</span><span class="st"> </span>.[[<span class="dv">1</span>]]</a> |
|
| 186 |
+<a class="sourceLine" id="cb9-8" data-line-number="8"> quer =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(ccdb<span class="op">$</span>contig_tbl, <span class="op">!!</span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/tidyeval.html">sym</a></span>(ccdb<span class="op">$</span>cluster_pk) <span class="op">==</span><span class="st"> </span>idx)</a> |
|
| 187 |
+<a class="sourceLine" id="cb9-9" data-line-number="9"> hc =<span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/stats/topics/hclust">hclust</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/stats/topics/dist">as.dist</a></span>(h<span class="op">$</span>distance_mat), <span class="dt">method =</span> method) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/ggdendro/topics/dendro_data">dendro_data</a></span>(<span class="dt">type =</span> <span class="st">"rectangle"</span>)</a> |
|
| 188 |
+<a class="sourceLine" id="cb9-10" data-line-number="10"> hc<span class="op">$</span>labels =<span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/cbind">cbind</a></span>(hc<span class="op">$</span>labels, quer)</a> |
|
| 189 |
+<a class="sourceLine" id="cb9-11" data-line-number="11"> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(hc<span class="op">$</span>segments, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x=</span>x, <span class="dt">y=</span>y)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_segment.html">geom_segment</a></span>(<span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">xend=</span>xend, <span class="dt">yend=</span>yend)) <span class="op">+</span><span class="st"> </span></a> |
|
| 190 |
+<a class="sourceLine" id="cb9-12" data-line-number="12"><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_classic</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_text.html">geom_text</a></span>(<span class="dt">data =</span> hc<span class="op">$</span>labels, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">color =</span> sample, <span class="dt">label =</span> fancy_name), <span class="dt">size =</span> <span class="dv">3</span>, <span class="dt">angle =</span> <span class="dv">60</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/scale_continuous.html">scale_x_continuous</a></span>(<span class="dt">breaks =</span> <span class="ot">NULL</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">ylab</a></span>(<span class="st">'AA Distance'</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">xlab</a></span>(<span class="st">''</span>)</a> |
|
| 191 |
+<a class="sourceLine" id="cb9-13" data-line-number="13">}</a> |
|
| 192 |
+<a class="sourceLine" id="cb9-14" data-line-number="14"></a> |
|
| 193 |
+<a class="sourceLine" id="cb9-15" data-line-number="15">MIN_OLIGO =<span class="st"> </span><span class="dv">7</span></a> |
|
| 194 |
+<a class="sourceLine" id="cb9-16" data-line-number="16">to_plot =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(aa80<span class="op">$</span>cluster_tbl, n_cluster <span class="op">>=</span><span class="st"> </span>MIN_OLIGO)</a> |
|
| 195 |
+<a class="sourceLine" id="cb9-17" data-line-number="17"></a> |
|
| 196 |
+<a class="sourceLine" id="cb9-18" data-line-number="18"><span class="kw"><a href="https://purrr.tidyverse.org/reference/map.html">map</a></span>(to_plot<span class="op">$</span>aa80, <span class="op">~</span><span class="st"> </span><span class="kw">dendro_plot</span>(aa80, .))</a> |
|
| 197 |
+<a class="sourceLine" id="cb9-19" data-line-number="19"><span class="co">#> [[1]]</span></a></code></pre></div> |
|
| 198 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-9-1.png" width="700"></p> |
|
| 199 |
+<pre><code>#> |
|
| 200 |
+#> [[2]]</code></pre> |
|
| 201 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-9-2.png" width="700"></p> |
|
| 202 |
+<pre><code>#> |
|
| 203 |
+#> [[3]]</code></pre> |
|
| 204 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-9-3.png" width="700"></p> |
|
| 205 |
+<pre><code>#> |
|
| 206 |
+#> [[4]]</code></pre> |
|
| 207 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-9-4.png" width="700"></p> |
|
| 208 |
+<div class="sourceCode" id="cb13"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb13-1" data-line-number="1">aa80 =<span class="st"> </span>CellaRepertorium<span class="op">:::</span><span class="kw">canonicalize_cluster</span>(aa80, <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'cdr3'</span>), <span class="dt">contig_fields =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'cdr3'</span>, <span class="st">'cdr3_nt'</span>, <span class="st">'chain'</span>, <span class="st">'v_gene'</span>, <span class="st">'d_gene'</span>, <span class="st">'j_gene'</span>))</a></code></pre></div> |
|
| 209 |
+<p>Pull the fields listed in <code>contig_fields</code> into the <code>cluster_tbl</code>, using the values found in the medoid contig</p> |
|
| 210 |
+<div class="sourceCode" id="cb14"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb14-1" data-line-number="1">oligo_clusters =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(aa80<span class="op">$</span>cluster_tbl, n_cluster <span class="op">>=</span><span class="st"> </span>MIN_OLIGO)</a> |
|
| 211 |
+<a class="sourceLine" id="cb14-2" data-line-number="2">oligo_contigs =<span class="st"> </span>aa80</a> |
|
| 212 |
+<a class="sourceLine" id="cb14-3" data-line-number="3">oligo_contigs<span class="op">$</span>contig_tbl =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">semi_join</a></span>(oligo_contigs<span class="op">$</span>contig_tbl, oligo_clusters, <span class="dt">by =</span> <span class="st">'aa80'</span>)</a> |
|
| 213 |
+<a class="sourceLine" id="cb14-4" data-line-number="4">oligo_contigs</a> |
|
| 214 |
+<a class="sourceLine" id="cb14-5" data-line-number="5"><span class="co">#> ContigCellDB of 54 contigs; 54 cells; and 4 clusters.</span></a> |
|
| 215 |
+<a class="sourceLine" id="cb14-6" data-line-number="6"><span class="co">#> Contigs keyed by barcode, pop, sample, contig_id; cells keyed by barcode, pop, sample.</span></a></code></pre></div> |
|
| 216 |
+<p>Get contigs/cells/clusters found at least 7 times (across contigs). Note that replacing <code>contig_tbl</code> with the subset selected with the <code>semi_join</code> also automatically subsetted the <code>cell_tbl</code> and <code>cluster_tbl</code>.</p> |
|
| 217 |
+<div class="sourceCode" id="cb15"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb15-1" data-line-number="1">oligo_clusters =<span class="st"> </span>oligo_contigs<span class="op">$</span>contig_tbl <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">group_by</a></span>(aa80) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/summarise.html">summarize</a></span>(<span class="st">`</span><span class="dt">n subjects observed</span><span class="st">`</span> =<span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/length">length</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/unique">unique</a></span>(sample))) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">left_join</a></span>(oligo_clusters)</a> |
|
| 218 |
+<a class="sourceLine" id="cb15-2" data-line-number="2"><span class="co">#> Joining, by = "aa80"</span></a> |
|
| 219 |
+<a class="sourceLine" id="cb15-3" data-line-number="3"></a> |
|
| 220 |
+<a class="sourceLine" id="cb15-4" data-line-number="4">knitr<span class="op">::</span><span class="kw"><a href="https://www.rdocumentation.org/packages/knitr/topics/kable">kable</a></span>(oligo_clusters <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/select.html">select</a></span>(aa80<span class="op">:</span>cdr3, chain<span class="op">:</span>j_gene, avg_distance, n_cluster))</a></code></pre></div> |
|
| 221 |
+<table class="table"> |
|
| 222 |
+<thead><tr class="header"> |
|
| 223 |
+<th align="right">aa80</th> |
|
| 224 |
+<th align="right">n subjects observed</th> |
|
| 225 |
+<th align="left">cdr3</th> |
|
| 226 |
+<th align="left">chain</th> |
|
| 227 |
+<th align="left">v_gene</th> |
|
| 228 |
+<th align="left">d_gene</th> |
|
| 229 |
+<th align="left">j_gene</th> |
|
| 230 |
+<th align="right">avg_distance</th> |
|
| 231 |
+<th align="right">n_cluster</th> |
|
| 232 |
+</tr></thead> |
|
| 233 |
+<tbody> |
|
| 234 |
+<tr class="odd"> |
|
| 235 |
+<td align="right">111</td> |
|
| 236 |
+<td align="right">6</td> |
|
| 237 |
+<td align="left">CVVGDRGSALGRLHF</td> |
|
| 238 |
+<td align="left">TRA</td> |
|
| 239 |
+<td align="left">TRAV11</td> |
|
| 240 |
+<td align="left">None</td> |
|
| 241 |
+<td align="left">TRAJ18</td> |
|
| 242 |
+<td align="right">0.6071429</td> |
|
| 243 |
+<td align="right">28</td> |
|
| 244 |
+</tr> |
|
| 245 |
+<tr class="even"> |
|
| 246 |
+<td align="right">172</td> |
|
| 247 |
+<td align="right">5</td> |
|
| 248 |
+<td align="left">CAVSRASSGSWQLIF</td> |
|
| 249 |
+<td align="left">TRA</td> |
|
| 250 |
+<td align="left">TRAV9N-3</td> |
|
| 251 |
+<td align="left">None</td> |
|
| 252 |
+<td align="left">TRAJ22</td> |
|
| 253 |
+<td align="right">2.1111111</td> |
|
| 254 |
+<td align="right">9</td> |
|
| 255 |
+</tr> |
|
| 256 |
+<tr class="odd"> |
|
| 257 |
+<td align="right">296</td> |
|
| 258 |
+<td align="right">6</td> |
|
| 259 |
+<td align="left">CAASASSGSWQLIF</td> |
|
| 260 |
+<td align="left">TRA</td> |
|
| 261 |
+<td align="left">TRAV14D-2</td> |
|
| 262 |
+<td align="left">None</td> |
|
| 263 |
+<td align="left">TRAJ22</td> |
|
| 264 |
+<td align="right">1.5000000</td> |
|
| 265 |
+<td align="right">8</td> |
|
| 266 |
+</tr> |
|
| 267 |
+<tr class="even"> |
|
| 268 |
+<td align="right">808</td> |
|
| 269 |
+<td align="right">4</td> |
|
| 270 |
+<td align="left">CATGNYAQGLTF</td> |
|
| 271 |
+<td align="left">TRA</td> |
|
| 272 |
+<td align="left">TRAV8D-2</td> |
|
| 273 |
+<td align="left">None</td> |
|
| 274 |
+<td align="left">TRAJ26</td> |
|
| 275 |
+<td align="right">1.3333333</td> |
|
| 276 |
+<td align="right">9</td> |
|
| 277 |
+</tr> |
|
| 278 |
+</tbody> |
|
| 279 |
+</table> |
|
| 280 |
+<p>Report some statistics about these expanded clusters.</p> |
|
| 281 |
+</div> |
|
| 282 |
+<div id="oligo-clusters" class="section level1"> |
|
| 283 |
+<h1 class="hasAnchor"> |
|
| 284 |
+<a href="#oligo-clusters" class="anchor"></a>Oligo clusters</h1> |
|
| 285 |
+<div class="sourceCode" id="cb16"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb16-1" data-line-number="1">oligo_plot =<span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(oligo_contigs<span class="op">$</span>contig_tbl, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> representative, <span class="dt">fill =</span> chain)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_bar.html">geom_bar</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/coord_flip.html">coord_flip</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/scale_brewer.html">scale_fill_brewer</a></span>(<span class="dt">type =</span> <span class="st">'qual'</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_minimal</a></span>()</a> |
|
| 286 |
+<a class="sourceLine" id="cb16-2" data-line-number="2">oligo_plot</a></code></pre></div> |
|
| 287 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-13-1.png" width="700"></p> |
|
| 288 |
+<p>These always come from a single chain.</p> |
|
| 289 |
+<div class="sourceCode" id="cb17"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb17-1" data-line-number="1">oligo_plot <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">fill =</span> sample) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/facet_wrap.html">facet_wrap</a></span>(<span class="op">~</span>pop)</a></code></pre></div> |
|
| 290 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-14-1.png" width="700"></p> |
|
| 291 |
+<p>But come from multiple populations and samples.</p> |
|
| 292 |
+</div> |
|
| 293 |
+<div id="formal-testing-for-frequency-differences" class="section level1"> |
|
| 294 |
+<h1 class="hasAnchor"> |
|
| 295 |
+<a href="#formal-testing-for-frequency-differences" class="anchor"></a>Formal testing for frequency differences</h1> |
|
| 296 |
+<div class="sourceCode" id="cb18"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb18-1" data-line-number="1"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(lme4)</a> |
|
| 297 |
+<a class="sourceLine" id="cb18-2" data-line-number="2"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(broom)</a> |
|
| 298 |
+<a class="sourceLine" id="cb18-3" data-line-number="3">per_chain_sample =<span class="st"> </span>good_cluster_cells <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">group_by</a></span>(sample, pop, chain) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/summarise.html">summarize</a></span>(<span class="dt">total_cells =</span> <span class="kw"><a href="https://dplyr.tidyverse.org/reference/n.html">n</a></span>(), <span class="dt">weeks_premature =</span> weeks_premature[<span class="dv">1</span>])</a> |
|
| 299 |
+<a class="sourceLine" id="cb18-4" data-line-number="4"></a> |
|
| 300 |
+<a class="sourceLine" id="cb18-5" data-line-number="5">oligo_cluster_stat =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">semi_join</a></span>(oligo_clusters, good_cluster_cells <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/select.html">select</a></span>(dataset, contig_id)) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">group_by</a></span>(sample, pop, chain, cluster_idx) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/summarise.html">summarize</a></span>(<span class="dt">n_cluster =</span> <span class="kw"><a href="https://dplyr.tidyverse.org/reference/n.html">n</a></span>())<span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">ungroup</a></span>() <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://tidyr.tidyverse.org/reference/complete.html">complete</a></span>(sample, pop, <span class="kw"><a href="https://tidyr.tidyverse.org/reference/expand.html">nesting</a></span>(cluster_idx, chain), <span class="dt">fill =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/list">list</a></span>(<span class="dt">n_cluster =</span> <span class="dv">0</span>))</a> |
|
| 301 |
+<a class="sourceLine" id="cb18-6" data-line-number="6"></a> |
|
| 302 |
+<a class="sourceLine" id="cb18-7" data-line-number="7">oligo_cluster_stat =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">left_join</a></span>(oligo_cluster_stat, per_chain_sample, <span class="dt">by =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'sample'</span>, <span class="st">'pop'</span>, <span class="st">'chain'</span>)) </a> |
|
| 303 |
+<a class="sourceLine" id="cb18-8" data-line-number="8"></a> |
|
| 304 |
+<a class="sourceLine" id="cb18-9" data-line-number="9"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/stopifnot">stopifnot</a></span>( <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/all">all</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/colSums">colSums</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/with">with</a></span>(oligo_cluster_stat, <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/table">table</a></span>(chain, cluster_idx)) <span class="op">></span><span class="st"> </span><span class="dv">0</span>) <span class="op">==</span><span class="st"> </span><span class="dv">1</span>))</a> |
|
| 305 |
+<a class="sourceLine" id="cb18-10" data-line-number="10"></a> |
|
| 306 |
+<a class="sourceLine" id="cb18-11" data-line-number="11">mm_out =<span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/warning">suppressWarnings</a></span>(oligo_cluster_stat <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">group_by</a></span>(cluster_idx, chain) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/do.html">do</a></span>( <span class="kw"><a href="https://www.rdocumentation.org/packages/lme4/topics/glmer">glmer</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/cbind">cbind</a></span>(n_cluster, total_cells) <span class="op">~</span><span class="st"> </span>pop <span class="op">+</span><span class="st"> </span>weeks_premature <span class="op">+</span><span class="st"> </span>(<span class="dv">1</span><span class="op">|</span>sample), <span class="dt">data =</span> ., <span class="dt">family =</span> <span class="st">'binomial'</span>) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/broom/topics/reexports">tidy</a></span>(<span class="dt">conf.int =</span> <span class="ot">TRUE</span>)))</a></code></pre></div> |
|
| 307 |
+<div class="sourceCode" id="cb19"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb19-1" data-line-number="1">mm_outj =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(<span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">left_join</a></span>(<span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">ungroup</a></span>(mm_out), <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/unique">unique</a></span>(oligo_clusters_all <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/select.html">select</a></span>(cdr3_representative, cluster_idx))), term <span class="op">%in%</span><span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'popCD31Pos'</span>, <span class="st">'weeks_premature'</span>)) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>(<span class="dt">ci_lo =</span> AMmisc<span class="op">::</span><span class="kw"><a href="https://www.rdocumentation.org/packages/AMmisc/topics/clamp">clamp</a></span>(conf.low), <span class="dt">ci_hi =</span> AMmisc<span class="op">::</span><span class="kw"><a href="https://www.rdocumentation.org/packages/AMmisc/topics/clamp">clamp</a></span>(conf.high)) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/arrange.html">arrange</a></span>(<span class="kw"><a href="https://dplyr.tidyverse.org/reference/desc.html">desc</a></span>(cdr3_representative))</a> |
|
| 308 |
+<a class="sourceLine" id="cb19-2" data-line-number="2"></a> |
|
| 309 |
+<a class="sourceLine" id="cb19-3" data-line-number="3"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(mm_outj, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> cdr3_representative, <span class="dt">ymin =</span> ci_lo, <span class="dt">ymax =</span> ci_hi, <span class="dt">y =</span> <span class="kw">clamp</span>(estimate))) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_linerange.html">geom_pointrange</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/facet_wrap.html">facet_wrap</a></span>(<span class="op">~</span>term, <span class="dt">scales =</span> <span class="st">'free'</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/coord_flip.html">coord_flip</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_minimal</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_abline.html">geom_hline</a></span>(<span class="dt">yintercept =</span> <span class="dv">0</span>, <span class="dt">lty =</span> <span class="dv">2</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">xlab</a></span>(<span class="st">"Isomorph"</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">ylab</a></span>(<span class="st">"log odds of isomorph"</span>)</a></code></pre></div> |
|
| 310 |
+<p>We test if the binomial rate of clone expression differs between CD31+/- or term, for each clone.</p> |
|
| 311 |
+</div> |
|
| 312 |
+<div id="clonal-pairs" class="section level1"> |
|
| 313 |
+<h1 class="hasAnchor"> |
|
| 314 |
+<a href="#clonal-pairs" class="anchor"></a>Clonal pairs</h1> |
|
| 315 |
+<div class="sourceCode" id="cb20"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb20-1" data-line-number="1">class_colors =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/reexports.html">data_frame</a></span>(<span class="dt">chain =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/unique">unique</a></span>(aa80<span class="op">$</span>cluster_tbl<span class="op">$</span>chain)) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>(<span class="dt">class_color =</span> RColorBrewer<span class="op">::</span><span class="kw"><a href="https://www.rdocumentation.org/packages/RColorBrewer/topics/ColorBrewer">brewer.pal</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/length">length</a></span>(chain),<span class="st">"Set1"</span>)[<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/seq">seq_along</a></span>(chain)])</a> |
|
| 316 |
+<a class="sourceLine" id="cb20-2" data-line-number="2"><span class="co">#> Warning: `data_frame()` is deprecated, use `tibble()`.</span></a> |
|
| 317 |
+<a class="sourceLine" id="cb20-3" data-line-number="3"><span class="co">#> This warning is displayed once per session.</span></a> |
|
| 318 |
+<a class="sourceLine" id="cb20-4" data-line-number="4"><span class="co">#> Warning in RColorBrewer::brewer.pal(length(chain), "Set1"): minimal value for n is 3, returning requested palette with 3 different levels</span></a> |
|
| 319 |
+<a class="sourceLine" id="cb20-5" data-line-number="5"></a> |
|
| 320 |
+<a class="sourceLine" id="cb20-6" data-line-number="6">aa80<span class="op">$</span>cluster_pk =<span class="st"> 'representative'</span></a> |
|
| 321 |
+<a class="sourceLine" id="cb20-7" data-line-number="7">pairing_list =<span class="st"> </span><span class="kw"><a href="../reference/pairing_tables.html">pairing_tables</a></span>(aa80, <span class="dt">table_order =</span> <span class="dv">2</span>, <span class="dt">orphan_level =</span> <span class="dv">1</span>, <span class="dt">min_expansion =</span> <span class="dv">2</span>, <span class="dt">cluster_keys =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'cdr3'</span>, <span class="st">'representative'</span>, <span class="st">'chain'</span>, <span class="st">'v_gene'</span>, <span class="st">'j_gene'</span>, <span class="st">'avg_distance'</span>))</a> |
|
| 322 |
+<a class="sourceLine" id="cb20-8" data-line-number="8"><span class="co">#> Warning: Factor `cluster_idx.2` contains implicit NA, consider using</span></a> |
|
| 323 |
+<a class="sourceLine" id="cb20-9" data-line-number="9"><span class="co">#> `forcats::fct_explicit_na`</span></a> |
|
| 324 |
+<a class="sourceLine" id="cb20-10" data-line-number="10"><span class="co">#> Warning: Column `representative` joining factors with different levels,</span></a> |
|
| 325 |
+<a class="sourceLine" id="cb20-11" data-line-number="11"><span class="co">#> coercing to character vector</span></a></code></pre></div> |
|
| 326 |
+<div class="sourceCode" id="cb21"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb21-1" data-line-number="1">pairs_plt =<span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(pairing_list<span class="op">$</span>cell_tbl, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> cluster_idx<span class="fl">.1</span>_fct, <span class="dt">y =</span> cluster_idx<span class="fl">.2</span>_fct, <span class="dt">color =</span> sample, <span class="dt">shape =</span> pop)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_jitter.html">geom_jitter</a></span>(<span class="dt">width =</span> <span class="fl">.3</span>, <span class="dt">height =</span> <span class="fl">.3</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_minimal</a></span>()</a> |
|
| 327 |
+<a class="sourceLine" id="cb21-2" data-line-number="2"></a> |
|
| 328 |
+<a class="sourceLine" id="cb21-3" data-line-number="3">ylab =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/reexports.html">data_frame</a></span>(<span class="dt">cdr3_representative =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot_build.html">ggplot_build</a></span>(pairs_plt)<span class="op">$</span>layout<span class="op">$</span>panel_params[[<span class="dv">1</span>]]<span class="op">$</span>y.label) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">left_join</a></span>(feature_tbl) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>(<span class="dt">class_color =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/ifelse">ifelse</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/NA">is.na</a></span>(class_color), <span class="st">'#E41A1C'</span>, class_color))</a> |
|
| 329 |
+<a class="sourceLine" id="cb21-4" data-line-number="4"></a> |
|
| 330 |
+<a class="sourceLine" id="cb21-5" data-line-number="5">xlab =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/reexports.html">data_frame</a></span>(<span class="dt">cdr3_representative =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot_build.html">ggplot_build</a></span>(pairs_plt)<span class="op">$</span>layout<span class="op">$</span>panel_params[[<span class="dv">1</span>]]<span class="op">$</span>x.label) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">left_join</a></span>(feature_tbl) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>(<span class="dt">class_color =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/ifelse">ifelse</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/NA">is.na</a></span>(class_color), <span class="st">'#E41A1C'</span>, class_color))</a> |
|
| 331 |
+<a class="sourceLine" id="cb21-6" data-line-number="6"></a> |
|
| 332 |
+<a class="sourceLine" id="cb21-7" data-line-number="7">pairs_plt =<span class="st"> </span>pairs_plt <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/theme.html">theme</a></span>(<span class="dt">axis.text.x =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/element.html">element_text</a></span>(<span class="dt">angle =</span> <span class="dv">90</span>, <span class="dt">color =</span> xlab<span class="op">$</span>class_color, <span class="dt">size =</span> <span class="dv">8</span>), <span class="dt">axis.text.y =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/element.html">element_text</a></span>(<span class="dt">color =</span> ylab<span class="op">$</span>class_color, <span class="dt">size =</span> <span class="dv">8</span>))</a> |
|
| 333 |
+<a class="sourceLine" id="cb21-8" data-line-number="8"></a> |
|
| 334 |
+<a class="sourceLine" id="cb21-9" data-line-number="9">pairs_plt</a></code></pre></div> |
|
| 335 |
+<div id="expanded-clones" class="section level2"> |
|
| 336 |
+<h2 class="hasAnchor"> |
|
| 337 |
+<a href="#expanded-clones" class="anchor"></a>Expanded clones</h2> |
|
| 338 |
+<div class="sourceCode" id="cb22"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb22-1" data-line-number="1">pairing_list =<span class="st"> </span><span class="kw"><a href="../reference/pairing_tables.html">pairing_tables</a></span>(oligo_clusters_all <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/select.html">select</a></span>(cdr3_representative, dataset, barcode, chain, umis, reads), <span class="dt">cluster_idx =</span> <span class="st">'cdr3_representative'</span>, <span class="dt">cell_identifiers =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'dataset'</span>, <span class="st">'barcode'</span>), <span class="dt">canonicalize_fun =</span> canonicalize_by_prevalence, <span class="dt">table_order =</span> <span class="dv">2</span>, <span class="dt">orphan_level =</span> <span class="dv">1</span>, <span class="dt">min_expansion =</span> <span class="dv">4</span>, <span class="dt">feature_tbl =</span> feature_tbl, <span class="dt">cell_tbl =</span> good_cells, <span class="dt">cluster_whitelist =</span> <span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(oligo_clusters, n_cluster<span class="op">></span><span class="dv">8</span>) <span class="op">%>%</span><span class="st"> </span>dplyr<span class="op">::</span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/select.html">select</a></span>(<span class="dt">cluster_idx.1 =</span> cdr3_representative) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/unique">unique</a></span>())</a> |
|
| 339 |
+<a class="sourceLine" id="cb22-2" data-line-number="2">pairs_plt =<span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(pairing_list<span class="op">$</span>cell_tbl, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> cluster_idx<span class="fl">.1</span>_fct, <span class="dt">y =</span> cluster_idx<span class="fl">.2</span>_fct, <span class="dt">color =</span> sample, <span class="dt">shape =</span> pop)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_jitter.html">geom_jitter</a></span>(<span class="dt">width =</span> <span class="fl">.3</span>, <span class="dt">height =</span> <span class="fl">.3</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_minimal</a></span>()</a> |
|
| 340 |
+<a class="sourceLine" id="cb22-3" data-line-number="3"></a> |
|
| 341 |
+<a class="sourceLine" id="cb22-4" data-line-number="4">ylab =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/reexports.html">data_frame</a></span>(<span class="dt">cdr3_representative =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot_build.html">ggplot_build</a></span>(pairs_plt)<span class="op">$</span>layout<span class="op">$</span>panel_params[[<span class="dv">1</span>]]<span class="op">$</span>y.label) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">left_join</a></span>(feature_tbl) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>(<span class="dt">class_color =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/ifelse">ifelse</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/NA">is.na</a></span>(class_color), <span class="st">'#E41A1C'</span>, class_color))</a> |
|
| 342 |
+<a class="sourceLine" id="cb22-5" data-line-number="5"></a> |
|
| 343 |
+<a class="sourceLine" id="cb22-6" data-line-number="6">xlab =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/reexports.html">data_frame</a></span>(<span class="dt">cdr3_representative =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot_build.html">ggplot_build</a></span>(pairs_plt)<span class="op">$</span>layout<span class="op">$</span>panel_params[[<span class="dv">1</span>]]<span class="op">$</span>x.label) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">left_join</a></span>(feature_tbl) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>(<span class="dt">class_color =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/ifelse">ifelse</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/NA">is.na</a></span>(class_color), <span class="st">'#E41A1C'</span>, class_color))</a> |
|
| 344 |
+<a class="sourceLine" id="cb22-7" data-line-number="7"></a> |
|
| 345 |
+<a class="sourceLine" id="cb22-8" data-line-number="8">pairs_plt =<span class="st"> </span>pairs_plt <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/theme.html">theme</a></span>(<span class="dt">axis.text.x =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/element.html">element_text</a></span>(<span class="dt">angle =</span> <span class="dv">90</span>, <span class="dt">color =</span> xlab<span class="op">$</span>class_color, <span class="dt">size =</span> <span class="dv">8</span>), <span class="dt">axis.text.y =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/element.html">element_text</a></span>(<span class="dt">color =</span> ylab<span class="op">$</span>class_color, <span class="dt">size =</span> <span class="dv">8</span>))</a> |
|
| 346 |
+<a class="sourceLine" id="cb22-9" data-line-number="9"></a> |
|
| 347 |
+<a class="sourceLine" id="cb22-10" data-line-number="10">pairs_plt</a></code></pre></div> |
|
| 348 |
+</div> |
|
| 349 |
+</div> |
|
| 350 |
+<div id="length-of-cdr3" class="section level1"> |
|
| 351 |
+<h1 class="hasAnchor"> |
|
| 352 |
+<a href="#length-of-cdr3" class="anchor"></a>Length of CDR3</h1> |
|
| 353 |
+<div class="sourceCode" id="cb23"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb23-1" data-line-number="1">aa80<span class="op">$</span>contig_tbl =<span class="st"> </span>aa80<span class="op">$</span>contig_tbl <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>(<span class="dt">cdr3_length =</span> <span class="kw"><a href="https://stringr.tidyverse.org/reference/str_length.html">str_length</a></span>(cdr3_nt))</a> |
|
| 354 |
+<a class="sourceLine" id="cb23-2" data-line-number="2"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(aa80<span class="op">$</span>contig_tbl, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">fill =</span> pop, <span class="dt">x=</span> cdr3_length)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_histogram.html">geom_histogram</a></span>(<span class="dt">binwidth =</span> <span class="dv">1</span>, <span class="dt">mapping =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">y =</span> ..density..)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_minimal</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/scale_brewer.html">scale_fill_brewer</a></span>(<span class="dt">type =</span> <span class="st">'qual'</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/facet_grid.html">facet_grid</a></span>(sample <span class="op">~</span>chain) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/theme.html">theme</a></span>(<span class="dt">strip.text.y =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/element.html">element_text</a></span>(<span class="dt">angle =</span> <span class="dv">0</span>)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/coord_cartesian.html">coord_cartesian</a></span>(<span class="dt">xlim =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="dv">25</span>, <span class="dv">55</span>))</a></code></pre></div> |
|
| 355 |
+<p><img src="cdr3_clustering_files/figure-html/unnamed-chunk-17-1.png" width="700"></p> |
|
| 356 |
+<p>Plot the CDR3 length distribution for each sample and pop. There doesn’t appear to be a noticable difference between Balbc and Black6 mice, but if we needed to make sure, an appropriate procedure would be to run a mixed model with a random <code>sample</code> effect (assumed to represent a biological replicate).</p> |
|
| 357 |
+<div class="sourceCode" id="cb24"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb24-1" data-line-number="1">cdr_len =<span class="st"> </span>aa80<span class="op">$</span>contig_tbl <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">group_by</a></span>(chain) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/do.html">do</a></span>(broom<span class="op">::</span><span class="kw"><a href="https://www.rdocumentation.org/packages/broom/topics/reexports">tidy</a></span>(lme4<span class="op">::</span><span class="kw"><a href="https://www.rdocumentation.org/packages/lme4/topics/lmer">lmer</a></span>(cdr3_length <span class="op">~</span><span class="st"> </span>pop <span class="op">+</span><span class="st"> </span>(<span class="dv">1</span><span class="op">|</span>sample), <span class="dt">data =</span> .), <span class="dt">conf.int =</span> <span class="ot">TRUE</span>))</a> |
|
| 358 |
+<a class="sourceLine" id="cb24-2" data-line-number="2"><span class="co">#> boundary (singular) fit: see ?isSingular</span></a> |
|
| 359 |
+<a class="sourceLine" id="cb24-3" data-line-number="3"><span class="co">#> Warning in bind_rows_(x, .id): binding factor and character vector,</span></a> |
|
| 360 |
+<a class="sourceLine" id="cb24-4" data-line-number="4"><span class="co">#> coercing into character vector</span></a> |
|
| 361 |
+<a class="sourceLine" id="cb24-5" data-line-number="5"><span class="co">#> Warning in bind_rows_(x, .id): binding character and factor vector,</span></a> |
|
| 362 |
+<a class="sourceLine" id="cb24-6" data-line-number="6"><span class="co">#> coercing into character vector</span></a> |
|
| 363 |
+<a class="sourceLine" id="cb24-7" data-line-number="7"><span class="co">#> boundary (singular) fit: see ?isSingular</span></a> |
|
| 364 |
+<a class="sourceLine" id="cb24-8" data-line-number="8"><span class="co">#> Warning in bind_rows_(x, .id): binding factor and character vector,</span></a> |
|
| 365 |
+<a class="sourceLine" id="cb24-9" data-line-number="9"><span class="co">#> coercing into character vector</span></a> |
|
| 366 |
+<a class="sourceLine" id="cb24-10" data-line-number="10"></a> |
|
| 367 |
+<a class="sourceLine" id="cb24-11" data-line-number="11"><span class="co">#> Warning in bind_rows_(x, .id): binding character and factor vector,</span></a> |
|
| 368 |
+<a class="sourceLine" id="cb24-12" data-line-number="12"><span class="co">#> coercing into character vector</span></a> |
|
| 369 |
+<a class="sourceLine" id="cb24-13" data-line-number="13"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(cdr_len <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(group <span class="op">==</span><span class="st"> 'fixed'</span>, term <span class="op">!=</span><span class="st"> '(Intercept)'</span>), <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/interaction">interaction</a></span>(chain, term), <span class="dt">y =</span> estimate, <span class="dt">ymin =</span> conf.low, <span class="dt">ymax =</span> conf.high)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_linerange.html">geom_pointrange</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_minimal</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/coord_flip.html">coord_flip</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">ylab</a></span>(<span class="st">'Length(CDR3 Nt)'</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">xlab</a></span>(<span class="st">'Term/Chain'</span>)</a></code></pre></div> |
|
| 370 |
+<p><img src="cdr3_clustering_files/figure-html/cdr3_len-1.png" width="288"></p> |
|
| 371 |
+<p>As was suggested by the histogram, there doesn’t seem to be an obvious <code>pop</code> effect.</p> |
|
| 372 |
+</div> |
|
| 373 |
+ </div> |
|
| 374 |
+ |
|
| 375 |
+ <div class="col-md-3 hidden-xs hidden-sm" id="sidebar"> |
|
| 376 |
+ <div id="tocnav"> |
|
| 377 |
+ <h2 class="hasAnchor"> |
|
| 378 |
+<a href="#tocnav" class="anchor"></a>Contents</h2> |
|
| 379 |
+ <ul class="nav nav-pills nav-stacked"> |
|
| 380 |
+<li><a href="#load-filtered-contig-files">Load filtered contig files</a></li> |
|
| 381 |
+ <li><a href="#chain-pairings">Chain pairings</a></li> |
|
| 382 |
+ <li><a href="#cluster-cdr3-protein-sequences">Cluster CDR3 protein sequences</a></li> |
|
| 383 |
+ <li><a href="#cluster-cdr3-dna-sequences">Cluster CDR3 DNA sequences</a></li> |
|
| 384 |
+ <li><a href="#oligo-clusters">Oligo clusters</a></li> |
|
| 385 |
+ <li><a href="#formal-testing-for-frequency-differences">Formal testing for frequency differences</a></li> |
|
| 386 |
+ <li> |
|
| 387 |
+<a href="#clonal-pairs">Clonal pairs</a><ul class="nav nav-pills nav-stacked"> |
|
| 388 |
+<li><a href="#expanded-clones">Expanded clones</a></li> |
|
| 389 |
+ </ul> |
|
| 390 |
+</li> |
|
| 391 |
+ <li><a href="#length-of-cdr3">Length of CDR3</a></li> |
|
| 392 |
+ </ul> |
|
| 393 |
+</div> |
|
| 394 |
+ </div> |
|
| 395 |
+ |
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+</div> |
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+ |
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+ |
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+ <footer><div class="copyright"> |
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+ <p>Developed by Andrew McDavid, Yu Gu.</p> |
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+</div> |
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+<div class="pkgdown"> |
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+ <p>Site built with <a href="https://pkgdown.r-lib.org/">pkgdown</a> 1.3.0.</p> |
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+ </footer> |
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+ <li><a href="cdr3_clustering.html">Clustering repertoire via CDR3 sequences</a></li> |
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+ <li><a href="mouse_tcell_qc.html">Quality control and Exploration of UMI-based repertoire data</a></li> |
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+ |
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+ </a> |
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+</li> |
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+ <a href="../reference/index.html">Reference</a> |
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+</li> |
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+<li class="dropdown"> |
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+ <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false"> |
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+ Articles |
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+ |
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+ <span class="caret"></span> |
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+ </a> |
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+ <ul class="dropdown-menu" role="menu"> |
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+<li> |
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+ <a href="../articles/cdr3_clustering.html">Clustering repertoire via CDR3 sequences</a> |
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+ </li> |
|
| 58 |
+ <li> |
|
| 59 |
+ <a href="../articles/mouse_tcell_qc.html">Quality control and Exploration of UMI-based repertoire data</a> |
|
| 60 |
+ </li> |
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+ </ul> |
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+</li> |
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+ </ul> |
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+<ul class="nav navbar-nav navbar-right"> |
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+ <span class="fa fa-github fa-lg"></span> |
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+ |
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+<!--/.nav-collapse --> |
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+<!--/.navbar --> |
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+ |
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+ |
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+ </header><div class="row"> |
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+ <div class="col-md-9 contents"> |
|
| 82 |
+ <div class="page-header toc-ignore"> |
|
| 83 |
+ <h1>Quality control and Exploration of UMI-based repertoire data</h1> |
|
| 84 |
+ |
|
| 85 |
+ |
|
| 86 |
+ <small class="dont-index">Source: <a href="https://github.com/amcdavid/CellaRepertorium/blob/master/vignettes/mouse_tcell_qc.Rmd"><code>vignettes/mouse_tcell_qc.Rmd</code></a></small> |
|
| 87 |
+ <div class="hidden name"><code>mouse_tcell_qc.Rmd</code></div> |
|
| 88 |
+ |
|
| 89 |
+ </div> |
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+ |
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+ |
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+ |
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| 93 |
+<div class="sourceCode" id="cb1"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb1-1" data-line-number="1"><span class="co">#load_all()</span></a> |
|
| 94 |
+<a class="sourceLine" id="cb1-2" data-line-number="2"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(CellaRepertorium)</a> |
|
| 95 |
+<a class="sourceLine" id="cb1-3" data-line-number="3"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(dplyr)</a> |
|
| 96 |
+<a class="sourceLine" id="cb1-4" data-line-number="4"><span class="co">#> </span></a> |
|
| 97 |
+<a class="sourceLine" id="cb1-5" data-line-number="5"><span class="co">#> Attaching package: 'dplyr'</span></a> |
|
| 98 |
+<a class="sourceLine" id="cb1-6" data-line-number="6"><span class="co">#> The following objects are masked from 'package:stats':</span></a> |
|
| 99 |
+<a class="sourceLine" id="cb1-7" data-line-number="7"><span class="co">#> </span></a> |
|
| 100 |
+<a class="sourceLine" id="cb1-8" data-line-number="8"><span class="co">#> filter, lag</span></a> |
|
| 101 |
+<a class="sourceLine" id="cb1-9" data-line-number="9"><span class="co">#> The following objects are masked from 'package:base':</span></a> |
|
| 102 |
+<a class="sourceLine" id="cb1-10" data-line-number="10"><span class="co">#> </span></a> |
|
| 103 |
+<a class="sourceLine" id="cb1-11" data-line-number="11"><span class="co">#> intersect, setdiff, setequal, union</span></a> |
|
| 104 |
+<a class="sourceLine" id="cb1-12" data-line-number="12"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(ggplot2)</a> |
|
| 105 |
+<a class="sourceLine" id="cb1-13" data-line-number="13"><span class="co">#> Registered S3 methods overwritten by 'ggplot2':</span></a> |
|
| 106 |
+<a class="sourceLine" id="cb1-14" data-line-number="14"><span class="co">#> method from </span></a> |
|
| 107 |
+<a class="sourceLine" id="cb1-15" data-line-number="15"><span class="co">#> [.quosures rlang</span></a> |
|
| 108 |
+<a class="sourceLine" id="cb1-16" data-line-number="16"><span class="co">#> c.quosures rlang</span></a> |
|
| 109 |
+<a class="sourceLine" id="cb1-17" data-line-number="17"><span class="co">#> print.quosures rlang</span></a> |
|
| 110 |
+<a class="sourceLine" id="cb1-18" data-line-number="18"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(readr)</a> |
|
| 111 |
+<a class="sourceLine" id="cb1-19" data-line-number="19"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(tidyr)</a> |
|
| 112 |
+<a class="sourceLine" id="cb1-20" data-line-number="20"><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/library">library</a></span>(stringr)</a></code></pre></div> |
|
| 113 |
+<div id="general-questions" class="section level1"> |
|
| 114 |
+<h1 class="hasAnchor"> |
|
| 115 |
+<a href="#general-questions" class="anchor"></a>General questions</h1> |
|
| 116 |
+<ol style="list-style-type: decimal"> |
|
| 117 |
+<li>Can/should elements of this script be included as package functionality (cell calling/filtering?)</li> |
|
| 118 |
+<li>Can the QC be made more statistically principaled?</li> |
|
| 119 |
+</ol> |
|
| 120 |
+</div> |
|
| 121 |
+<div id="load-contig-files" class="section level1"> |
|
| 122 |
+<h1 class="hasAnchor"> |
|
| 123 |
+<a href="#load-contig-files" class="anchor"></a>Load contig files</h1> |
|
| 124 |
+<div class="sourceCode" id="cb2"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb2-1" data-line-number="1">files =<span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/list.files">list.files</a></span>(<span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/system.file">system.file</a></span>(<span class="st">'extdata'</span>, <span class="dt">package =</span> <span class="st">'CellaRepertorium'</span>), <span class="dt">pattern =</span> <span class="st">"all_contig_annotations_.+?.csv.xz"</span>, <span class="dt">recursive =</span> <span class="ot">TRUE</span>, <span class="dt">full.names =</span> <span class="ot">TRUE</span>)</a> |
|
| 125 |
+<a class="sourceLine" id="cb2-2" data-line-number="2"><span class="co"># Pull out sample and population names</span></a> |
|
| 126 |
+<a class="sourceLine" id="cb2-3" data-line-number="3">samp_map =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/reexports.html">tibble</a></span>(<span class="dt">anno_file =</span> files, <span class="dt">pop =</span> <span class="kw"><a href="https://stringr.tidyverse.org/reference/str_match.html">str_match</a></span>(files, <span class="st">'b6|balbc'</span>)[,<span class="dv">1</span>], <span class="dt">sample =</span> <span class="kw"><a href="https://stringr.tidyverse.org/reference/str_match.html">str_match</a></span>(files, <span class="st">'_([0-9])</span><span class="ch">\\</span><span class="st">.'</span>)[,<span class="dv">2</span>])</a> |
|
| 127 |
+<a class="sourceLine" id="cb2-4" data-line-number="4"></a> |
|
| 128 |
+<a class="sourceLine" id="cb2-5" data-line-number="5">knitr<span class="op">::</span><span class="kw"><a href="https://www.rdocumentation.org/packages/knitr/topics/kable">kable</a></span>(samp_map)</a></code></pre></div> |
|
| 129 |
+<table class="table"> |
|
| 130 |
+<thead><tr class="header"> |
|
| 131 |
+<th align="left">anno_file</th> |
|
| 132 |
+<th align="left">pop</th> |
|
| 133 |
+<th align="left">sample</th> |
|
| 134 |
+</tr></thead> |
|
| 135 |
+<tbody> |
|
| 136 |
+<tr class="odd"> |
|
| 137 |
+<td align="left">/Users/amcdavid/Library/R/3.9-bioc/CellaRepertorium/extdata/all_contig_annotations_b6_4.csv.xz</td> |
|
| 138 |
+<td align="left">b6</td> |
|
| 139 |
+<td align="left">4</td> |
|
| 140 |
+</tr> |
|
| 141 |
+<tr class="even"> |
|
| 142 |
+<td align="left">/Users/amcdavid/Library/R/3.9-bioc/CellaRepertorium/extdata/all_contig_annotations_b6_5.csv.xz</td> |
|
| 143 |
+<td align="left">b6</td> |
|
| 144 |
+<td align="left">5</td> |
|
| 145 |
+</tr> |
|
| 146 |
+<tr class="odd"> |
|
| 147 |
+<td align="left">/Users/amcdavid/Library/R/3.9-bioc/CellaRepertorium/extdata/all_contig_annotations_b6_6.csv.xz</td> |
|
| 148 |
+<td align="left">b6</td> |
|
| 149 |
+<td align="left">6</td> |
|
| 150 |
+</tr> |
|
| 151 |
+<tr class="even"> |
|
| 152 |
+<td align="left">/Users/amcdavid/Library/R/3.9-bioc/CellaRepertorium/extdata/all_contig_annotations_balbc_1.csv.xz</td> |
|
| 153 |
+<td align="left">balbc</td> |
|
| 154 |
+<td align="left">1</td> |
|
| 155 |
+</tr> |
|
| 156 |
+<tr class="odd"> |
|
| 157 |
+<td align="left">/Users/amcdavid/Library/R/3.9-bioc/CellaRepertorium/extdata/all_contig_annotations_balbc_2.csv.xz</td> |
|
| 158 |
+<td align="left">balbc</td> |
|
| 159 |
+<td align="left">2</td> |
|
| 160 |
+</tr> |
|
| 161 |
+<tr class="even"> |
|
| 162 |
+<td align="left">/Users/amcdavid/Library/R/3.9-bioc/CellaRepertorium/extdata/all_contig_annotations_balbc_3.csv.xz</td> |
|
| 163 |
+<td align="left">balbc</td> |
|
| 164 |
+<td align="left">3</td> |
|
| 165 |
+</tr> |
|
| 166 |
+</tbody> |
|
| 167 |
+</table> |
|
| 168 |
+<p>PBMC pooled from Balb/C and C57BL/6 mice <a href="https://support.10xgenomics.com/single-cell-vdj/datasets/3.0.0/vdj_v1_mm_c57bl6_pbmc_t">were assayed on 10X genomics V3 chemistry</a> and a library enriched for TCR were run. For the purposes of illustrating functionality in this package, cell barcodes were subsampled 3 times for each of the Balb/C and Black6 pools to generate distinct <code>samples</code>, which is reflected in the <code>sample</code> column. More details are available in the scripts in the <code>data-raw</code> directory of this package.</p> |
|
| 169 |
+<div class="sourceCode" id="cb3"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb3-1" data-line-number="1"><span class="co"># read in CSV</span></a> |
|
| 170 |
+<a class="sourceLine" id="cb3-2" data-line-number="2">all_anno =<span class="st"> </span>samp_map <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/rowwise.html">rowwise</a></span>() <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>(<span class="dt">anno =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/list">list</a></span>(<span class="kw"><a href="https://readr.tidyverse.org/reference/read_delim.html">read_csv</a></span>(anno_file, <span class="dt">col_types =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/cols.html">cols</a></span>(</a> |
|
| 171 |
+<a class="sourceLine" id="cb3-3" data-line-number="3"> <span class="dt">barcode =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 172 |
+<a class="sourceLine" id="cb3-4" data-line-number="4"> <span class="dt">is_cell =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_logical</a></span>(),</a> |
|
| 173 |
+<a class="sourceLine" id="cb3-5" data-line-number="5"> <span class="dt">contig_id =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 174 |
+<a class="sourceLine" id="cb3-6" data-line-number="6"> <span class="dt">high_confidence =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_logical</a></span>(),</a> |
|
| 175 |
+<a class="sourceLine" id="cb3-7" data-line-number="7"> <span class="dt">length =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_double</a></span>(),</a> |
|
| 176 |
+<a class="sourceLine" id="cb3-8" data-line-number="8"> <span class="dt">chain =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 177 |
+<a class="sourceLine" id="cb3-9" data-line-number="9"> <span class="dt">v_gene =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 178 |
+<a class="sourceLine" id="cb3-10" data-line-number="10"> <span class="dt">d_gene =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 179 |
+<a class="sourceLine" id="cb3-11" data-line-number="11"> <span class="dt">j_gene =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 180 |
+<a class="sourceLine" id="cb3-12" data-line-number="12"> <span class="dt">c_gene =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 181 |
+<a class="sourceLine" id="cb3-13" data-line-number="13"> <span class="dt">full_length =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_logical</a></span>(),</a> |
|
| 182 |
+<a class="sourceLine" id="cb3-14" data-line-number="14"> <span class="dt">productive =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 183 |
+<a class="sourceLine" id="cb3-15" data-line-number="15"> <span class="dt">cdr3 =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 184 |
+<a class="sourceLine" id="cb3-16" data-line-number="16"> <span class="dt">cdr3_nt =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 185 |
+<a class="sourceLine" id="cb3-17" data-line-number="17"> <span class="dt">reads =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_double</a></span>(),</a> |
|
| 186 |
+<a class="sourceLine" id="cb3-18" data-line-number="18"> <span class="dt">umis =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_double</a></span>(),</a> |
|
| 187 |
+<a class="sourceLine" id="cb3-19" data-line-number="19"> <span class="dt">raw_clonotype_id =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>(),</a> |
|
| 188 |
+<a class="sourceLine" id="cb3-20" data-line-number="20"> <span class="dt">raw_consensus_id =</span> <span class="kw"><a href="https://readr.tidyverse.org/reference/parse_atomic.html">col_character</a></span>()</a> |
|
| 189 |
+<a class="sourceLine" id="cb3-21" data-line-number="21">))))</a> |
|
| 190 |
+<a class="sourceLine" id="cb3-22" data-line-number="22"></a> |
|
| 191 |
+<a class="sourceLine" id="cb3-23" data-line-number="23">all_anno =<span class="st"> </span>all_anno <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://tidyr.tidyverse.org/reference/unnest.html">unnest</a></span>() <span class="op">%>%</span><span class="st"> </span></a> |
|
| 192 |
+<a class="sourceLine" id="cb3-24" data-line-number="24"><span class="st"> </span><span class="co"># write a column specifying what cell type the contig belonged to</span></a> |
|
| 193 |
+<a class="sourceLine" id="cb3-25" data-line-number="25"><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/mutate.html">mutate</a></span>(<span class="dt">celltype =</span> <span class="kw"><a href="https://dplyr.tidyverse.org/reference/case_when.html">case_when</a></span>(chain <span class="op">%in%</span><span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'TRA'</span>, <span class="st">'TRB'</span>) <span class="op">~</span><span class="st"> "T_ab"</span>, chain <span class="op">%in%</span><span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'TRD'</span>, <span class="st">'TRG'</span>) <span class="op">~</span><span class="st"> 'T_gd'</span>, chain <span class="op">==</span><span class="st"> 'Multi'</span> <span class="op">~</span><span class="st"> 'Multi'</span>, chain <span class="op">%in%</span><span class="st"> </span><span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="st">'IGH'</span>,<span class="st">'IGK'</span>, <span class="st">'IGL'</span>) <span class="op">~</span><span class="st"> 'B'</span>, <span class="ot">TRUE</span> <span class="op">~</span><span class="st"> </span><span class="ot">NA_character_</span>))</a></code></pre></div> |
|
| 194 |
+<p>The pipeline for assembling reads into contigs, and mapping them to UMIs and cells is described in <a href="https://support.10xgenomics.com/single-cell-vdj/software/pipelines/latest/algorithms/overview">the 10X genomics documentation</a>, and its source code is available <a href="https://github.com/10XGenomics/cellranger/tree/5f5a6293bbc067e1965e50f0277286914b96c908">here</a>.</p> |
|
| 195 |
+<p>[Comment in greater depth on what output we are using, maybe make a schematic?]</p> |
|
| 196 |
+<p>We read in the contig annotation file for each of the samples, and annotate the contig as a alpha-beta T cell, gamma-delta T cell, B cell or chimeric “multi” cell type based on where various</p> |
|
| 197 |
+</div> |
|
| 198 |
+<div id="high-confidence-umis-belonging-to-t-cells-per-cell" class="section level1"> |
|
| 199 |
+<h1 class="hasAnchor"> |
|
| 200 |
+<a href="#high-confidence-umis-belonging-to-t-cells-per-cell" class="anchor"></a>High confidence UMIs belonging to T cells per cell</h1> |
|
| 201 |
+<div class="sourceCode" id="cb4"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb4-1" data-line-number="1">total_umi =<span class="st"> </span>all_anno <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">group_by</a></span>(sample, pop, barcode, is_cell, high_confidence, celltype <span class="op">==</span><span class="st"> 'T_ab'</span>) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/summarise.html">summarize</a></span>(<span class="dt">total_umi =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/sum">sum</a></span>(umis)) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/select.html">rename</a></span>(<span class="dt">is_T =</span> <span class="st">`</span><span class="dt">celltype == "T_ab"</span><span class="st">`</span>)</a> |
|
| 202 |
+<a class="sourceLine" id="cb4-2" data-line-number="2"></a> |
|
| 203 |
+<a class="sourceLine" id="cb4-3" data-line-number="3"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(<span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(total_umi, high_confidence, is_T), <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">color =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/factor">factor</a></span>(is_cell), <span class="dt">x =</span> total_umi, <span class="dt">group =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/interaction">interaction</a></span>(is_cell, sample, pop))) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/stat_ecdf.html">stat_ecdf</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/coord_cartesian.html">coord_cartesian</a></span>(<span class="dt">xlim =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/c">c</a></span>(<span class="dv">0</span>, <span class="dv">10</span>)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">ylab</a></span>(<span class="st">'Fraction of barcodes'</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_minimal</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/scale_hue.html">scale_color_discrete</a></span>(<span class="st">'10X called cell?'</span>)</a></code></pre></div> |
|
| 204 |
+<p><img src="mouse_tcell_qc_files/figure-html/unnamed-chunk-4-1.png" width="700"></p> |
|
| 205 |
+<p>10X defines <a href="https://support.10xgenomics.com/single-cell-vdj/software/pipelines/latest/algorithms/cell-calling">a procedure</a> to separate cells from background that fits a Gaussian mixture model to the UMI distributions for each sample. However in saome cases, it may be desirable to implement a common QC threshold with a different stringency, such as:</p> |
|
| 206 |
+<ul> |
|
| 207 |
+<li>Comparing across multiple samples</li> |
|
| 208 |
+<li>When a sample has been enriched for a particular cell type (eg with pre-sequencing flow cytometry).</li> |
|
| 209 |
+</ul> |
|
| 210 |
+<p>When we consider only high confidence UMIs that unambiguous map to T cells, most “non cells” have 1 or fewer, while most putative cells have >5. However, we might want to adopt a different UMI-based cell filter, as was done below. Is there a way to evaluate a sensitivity/specificity in distinguishing cells from debris, or T cells from other cell types?</p> |
|
| 211 |
+</div> |
|
| 212 |
+<div id="reads-umis" class="section level1"> |
|
| 213 |
+<h1 class="hasAnchor"> |
|
| 214 |
+<a href="#reads-umis" class="anchor"></a>Reads / UMIs</h1> |
|
| 215 |
+<div class="sourceCode" id="cb5"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb5-1" data-line-number="1">qual_plot =<span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(all_anno, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> celltype, <span class="dt">y=</span> umis)) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_violin.html">geom_violin</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_jitter.html">geom_jitter</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/facet_wrap.html">facet_wrap</a></span>(<span class="op">~</span>sample <span class="op">+</span><span class="st"> </span>pop) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/scale_continuous.html">scale_y_log10</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">xlab</a></span>(<span class="st">"Annotated cell type"</span>)</a> |
|
| 216 |
+<a class="sourceLine" id="cb5-2" data-line-number="2"></a> |
|
| 217 |
+<a class="sourceLine" id="cb5-3" data-line-number="3">qual_plot </a></code></pre></div> |
|
| 218 |
+<p><img src="mouse_tcell_qc_files/figure-html/unnamed-chunk-5-1.png" width="700"></p> |
|
| 219 |
+<div class="sourceCode" id="cb6"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb6-1" data-line-number="1">qual_plot <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">y =</span> reads)</a></code></pre></div> |
|
| 220 |
+<p><img src="mouse_tcell_qc_files/figure-html/unnamed-chunk-5-2.png" width="700"></p> |
|
| 221 |
+<p>The number of UMIs and reads by sample and annotated cell type.</p> |
|
| 222 |
+</div> |
|
| 223 |
+<div id="apply-t-cell-contig-umi-filter" class="section level1"> |
|
| 224 |
+<h1 class="hasAnchor"> |
|
| 225 |
+<a href="#apply-t-cell-contig-umi-filter" class="anchor"></a>Apply T-cell contig UMI filter</h1> |
|
| 226 |
+<div class="sourceCode" id="cb7"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb7-1" data-line-number="1"><span class="co"># At least 2 UMI mapping to high confidence T cell contigs.</span></a> |
|
| 227 |
+<a class="sourceLine" id="cb7-2" data-line-number="2">good_bc =<span class="st"> </span>total_umi <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">ungroup</a></span>() <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(is_cell) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(total_umi <span class="op">>=</span><span class="st"> </span><span class="dv">2</span>, is_T) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(high_confidence)</a> |
|
| 228 |
+<a class="sourceLine" id="cb7-3" data-line-number="3">total_cells =<span class="st"> </span>good_bc <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">group_by</a></span>(sample, pop) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/summarise.html">summarize</a></span>(<span class="dt">good_bc =</span> <span class="kw"><a href="https://dplyr.tidyverse.org/reference/n.html">n</a></span>())</a> |
|
| 229 |
+<a class="sourceLine" id="cb7-4" data-line-number="4">knitr<span class="op">::</span><span class="kw"><a href="https://www.rdocumentation.org/packages/knitr/topics/kable">kable</a></span>(total_cells)</a></code></pre></div> |
|
| 230 |
+<table class="table"> |
|
| 231 |
+<thead><tr class="header"> |
|
| 232 |
+<th align="left">sample</th> |
|
| 233 |
+<th align="left">pop</th> |
|
| 234 |
+<th align="right">good_bc</th> |
|
| 235 |
+</tr></thead> |
|
| 236 |
+<tbody> |
|
| 237 |
+<tr class="odd"> |
|
| 238 |
+<td align="left">1</td> |
|
| 239 |
+<td align="left">balbc</td> |
|
| 240 |
+<td align="right">133</td> |
|
| 241 |
+</tr> |
|
| 242 |
+<tr class="even"> |
|
| 243 |
+<td align="left">2</td> |
|
| 244 |
+<td align="left">balbc</td> |
|
| 245 |
+<td align="right">137</td> |
|
| 246 |
+</tr> |
|
| 247 |
+<tr class="odd"> |
|
| 248 |
+<td align="left">3</td> |
|
| 249 |
+<td align="left">balbc</td> |
|
| 250 |
+<td align="right">143</td> |
|
| 251 |
+</tr> |
|
| 252 |
+<tr class="even"> |
|
| 253 |
+<td align="left">4</td> |
|
| 254 |
+<td align="left">b6</td> |
|
| 255 |
+<td align="right">149</td> |
|
| 256 |
+</tr> |
|
| 257 |
+<tr class="odd"> |
|
| 258 |
+<td align="left">5</td> |
|
| 259 |
+<td align="left">b6</td> |
|
| 260 |
+<td align="right">150</td> |
|
| 261 |
+</tr> |
|
| 262 |
+<tr class="even"> |
|
| 263 |
+<td align="left">6</td> |
|
| 264 |
+<td align="left">b6</td> |
|
| 265 |
+<td align="right">148</td> |
|
| 266 |
+</tr> |
|
| 267 |
+</tbody> |
|
| 268 |
+</table> |
|
| 269 |
+<p>Apply a filter on UMIs.</p> |
|
| 270 |
+<div class="sourceCode" id="cb8"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb8-1" data-line-number="1">contigs_qc =<span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/join.html">semi_join</a></span>(all_anno, good_bc <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/select.html">select</a></span>(sample, pop, barcode)) <span class="op">%>%</span><span class="st"> </span></a> |
|
| 271 |
+<a class="sourceLine" id="cb8-2" data-line-number="2"><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/filter.html">filter</a></span>(full_length, productive <span class="op">==</span><span class="st"> 'True'</span>, high_confidence, chain <span class="op">!=</span><span class="st"> 'Multi'</span>)</a> |
|
| 272 |
+<a class="sourceLine" id="cb8-3" data-line-number="3"><span class="co">#> Joining, by = c("pop", "sample", "barcode")</span></a></code></pre></div>
|
|
| 273 |
+<p>And take only high confidence, full length, productive <span class="math inline">\(\alpha-\beta\)</span> T cell contigs.</p> |
|
| 274 |
+</div> |
|
| 275 |
+<div id="multi-chain-t-cells" class="section level1"> |
|
| 276 |
+<h1 class="hasAnchor"> |
|
| 277 |
+<a href="#multi-chain-t-cells" class="anchor"></a>“Multi-chain” T cells</h1> |
|
| 278 |
+<div class="sourceCode" id="cb9"><pre class="sourceCode r"><code class="sourceCode r"><a class="sourceLine" id="cb9-1" data-line-number="1"></a> |
|
| 279 |
+<a class="sourceLine" id="cb9-2" data-line-number="2">n_productive =<span class="st"> </span>contigs_qc <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/group_by.html">group_by</a></span>(sample, pop, barcode) <span class="op">%>%</span><span class="st"> </span><span class="kw"><a href="https://dplyr.tidyverse.org/reference/summarise.html">summarize</a></span>(<span class="dt">n_productive =</span> <span class="kw"><a href="https://dplyr.tidyverse.org/reference/n.html">n</a></span>())</a> |
|
| 280 |
+<a class="sourceLine" id="cb9-3" data-line-number="3"></a> |
|
| 281 |
+<a class="sourceLine" id="cb9-4" data-line-number="4"><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggplot.html">ggplot</a></span>(n_productive, <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/aes.html">aes</a></span>(<span class="dt">x =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/interaction">interaction</a></span>(sample,pop), <span class="dt">fill =</span> <span class="kw"><a href="https://www.rdocumentation.org/packages/base/topics/factor">factor</a></span>(n_productive))) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/geom_bar.html">geom_bar</a></span>(<span class="dt">position =</span> <span class="kw"><a href="https://ggplot2.tidyverse.org/reference/position_stack.html">position_stack</a></span>()) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/ggtheme.html">theme_minimal</a></span>() <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">xlab</a></span>(<span class="st">"Replicate"</span>) <span class="op">+</span><span class="st"> </span><span class="kw"><a href="https://ggplot2.tidyverse.org/reference/labs.html">ylab</a></span>(<span class="st">"Number of cell barcodes"</span>)</a></code></pre></div> |
|
| 282 |
+<p><img src="mouse_tcell_qc_files/figure-html/unnamed-chunk-8-1.png" width="700"></p> |
|
| 283 |
+<p>Number of productive chains per replicate.</p> |
|
| 284 |
+</div> |
|
| 285 |
+<div id="add-a-plot-showing-chain-breakdowns-alpha-alphaalpha-beta-alpha-alpha-beta-etc" class="section level1"> |
|
| 286 |
+<h1 class="hasAnchor"> |
|
| 287 |
+<a href="#add-a-plot-showing-chain-breakdowns-alpha-alphaalpha-beta-alpha-alpha-beta-etc" class="anchor"></a>Add a plot showing chain breakdowns alpha-alpha/alpha-beta, alpha-alpha-beta, etc</h1> |
|
| 288 |
+</div> |
|
| 289 |
+ </div> |
|
| 290 |
+ |
|
| 291 |
+ <div class="col-md-3 hidden-xs hidden-sm" id="sidebar"> |
|
| 292 |
+ <div id="tocnav"> |
|
| 293 |
+ <h2 class="hasAnchor"> |
|
| 294 |
+<a href="#tocnav" class="anchor"></a>Contents</h2> |
|
| 295 |
+ <ul class="nav nav-pills nav-stacked"> |
|
| 296 |
+<li><a href="#general-questions">General questions</a></li> |
|
| 297 |
+ <li><a href="#load-contig-files">Load contig files</a></li> |
|
| 298 |
+ <li><a href="#high-confidence-umis-belonging-to-t-cells-per-cell">High confidence UMIs belonging to T cells per cell</a></li> |
|
| 299 |
+ <li><a href="#reads-umis">Reads / UMIs</a></li> |
|
| 300 |
+ <li><a href="#apply-t-cell-contig-umi-filter">Apply T-cell contig UMI filter</a></li> |
|
| 301 |
+ <li><a href="#multi-chain-t-cells">“Multi-chain” T cells</a></li> |
|
| 302 |
+ <li><a href="#add-a-plot-showing-chain-breakdowns-alpha-alphaalpha-beta-alpha-alpha-beta-etc">Add a plot showing chain breakdowns alpha-alpha/alpha-beta, alpha-alpha-beta, etc</a></li> |
|
| 303 |
+ </ul> |
|
| 304 |
+</div> |
|
| 305 |
+ </div> |
|
| 306 |
+ |
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| 307 |
+</div> |
|
| 308 |
+ |
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| 309 |
+ |
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| 310 |
+ <footer><div class="copyright"> |
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+ <p>Developed by Andrew McDavid, Yu Gu.</p> |
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+</div> |
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+ |
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+<div class="pkgdown"> |
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+ <p>Site built with <a href="https://pkgdown.r-lib.org/">pkgdown</a> 1.3.0.</p> |
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+</div> |
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+ </footer> |
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+</div> |
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+</html> |
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+ </button> |
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+ <span class="navbar-brand"> |
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+ <a class="navbar-link" href="index.html">CellaRepertorium</a> |
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+ |
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+ </a> |
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+</li> |
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+<li> |
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+ <a href="reference/index.html">Reference</a> |
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+</li> |
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+<li class="dropdown"> |
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+ <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false"> |
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+ Articles |
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+ <li> |
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+ <a href="articles/cdr3_clustering.html">Clustering repertoire via CDR3 sequences</a> |
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+ </li> |
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+ <li> |
|
| 89 |
+ <a href="articles/mouse_tcell_qc.html">Quality control and Exploration of UMI-based repertoire data</a> |
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+ </li> |
|
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+ </ul> |
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+</li> |
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+ </ul> |
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+ |
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+ <ul class="nav navbar-nav navbar-right"> |
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+ <span class="fa fa-github fa-lg"></span> |
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+ <div class="contents col-md-9"> |
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+ <div class="page-header"> |
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+ <h1>Authors</h1> |
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+ |
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+ <ul class="list-unstyled"> |
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+ <li> |
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+ <p><strong>Andrew McDavid</strong>. Author, maintainer. |
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+ </p> |
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+ </li> |
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+ <li> |
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| 123 |
+ <p><strong>Yu Gu</strong>. Author. |
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+ </p> |
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+ </ul> |
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+ |
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+ <p>Developed by Andrew McDavid, Yu Gu.</p> |
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+</div> |
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+ |
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+ <p>Site built with <a href="https://pkgdown.r-lib.org/">pkgdown</a> 1.3.0.</p> |
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