From 7cd3be55cd67566d23d9272600e484edc72edbf8 Mon Sep 17 00:00:00 2001 From: Egor Dolzhenko Date: Fri, 14 Jan 2022 10:38:33 -0800 Subject: [PATCH] Add metrics docs (#41) * Add metrics intro --- README.md | 76 +++++++++++---------------- docs/images/allele-depth-example.png | Bin 0 -> 20191 bytes docs/images/intro-example.png | Bin 0 -> 254567 bytes docs/method-overview.md | 43 +++++++++++++++ docs/metrics.md | 26 +++++++++ 5 files changed, 99 insertions(+), 46 deletions(-) create mode 100644 docs/images/allele-depth-example.png create mode 100644 docs/images/intro-example.png create mode 100644 docs/method-overview.md create mode 100644 docs/metrics.md diff --git a/README.md b/README.md index 26d3256f1..0edc69ebf 100644 --- a/README.md +++ b/README.md @@ -5,15 +5,22 @@ tandem repeats. REViewer requires a BAMlet with graph-realigned reads generated by [ExpansionHunter](https://github.com/Illumina/ExpansionHunter) and the corresponding variant catalog. +![Introductory example](docs/images/intro-example.png) + ## License REViewer is provided under the terms and conditions of the [GPLv3 license](LICENSE.txt). It relies on several third party packages provided under other open source licenses, please see [COPYRIGHT.txt](COPYRIGHT.txt) for additional details. -## Building +## Installation + +The simplest way of obtaining REViewer is by downloading a Linux binary +corresponding to the latest release from the +[Releases page](https://github.com/Illumina/REViewer/releases). The link to the +binary is located in the *Assets* section. -REViewer can be built from source using the standard CMake commands. +REViewer can also be built from source with CMake. ```shell script cd REViewer @@ -38,56 +45,33 @@ REViewer \ --output-prefix ``` -Note that the BAMlet generated by ExpansionHunter (`--reads` parameter) must be sorted and indexed. - -## Examples of read pileups - -[This document](docs/examples.md) describes read pileups corresponding to correctly and incorrectly -genotyped repeats. It is a good starting point for learning how to use the program. +Note that the BAMlet generated by ExpansionHunter (`--reads` parameter) must be +sorted and indexed. -## Companion tool +## Introductory guides -[reviewer #2](https://github.com/broadinstitute/reviewer2) is a companion tool -for REViewer developed by [Ben Weisburd](https://github.com/bw2) that provides a -convenient way to assess large quantities of read pileups. - -## Overview of the method +- [A blog post describing the method](https://www.illumina.com/science/genomics-research/reviewer-visualizing-alignments-short-reads-long-repeat.html) +- [Examples of read pileups](docs/examples.md) corresponding to correctly and +incorrectly genotyped repeats -REViewer is designed to display alignments of reads generated by ExpansionHunter (Figure 3, -boxes 1-3). These alignments are obtained by realigning reads originating in the target region -to the corresponding sequence graph encoding one or more repeats located there5. REViewer then -constructs putative haplotype sequences using genotypes produced by ExpansionHunter and then -selects a pair of haplotypes that have the highest consistency with the read alignments (Figure 3, -boxes 4-6). (This step is skipped for repeats on haploid chromosomes.) Next, REViewer determines -the set of possible alignment positions for each read pair on each haplotype. For example, a read -pair originating within a flanking sequence shared by both haplotypes has exactly one alignment -position on each haplotype (Figure 3, Box 7a) while a read pair whose both mates are comprised -of the repeat sequence has multiple possible origins on haplotypes with sufficiently long repeats -(Figure 3, Box 7b). To generate a read pileup, REViewer selects one alignment position at random -for each read pair. This step is repeated a specified number of times (10,000 by default) to -generate multiple pileups. The pileup with the most even coverage of each allele is selected -for visualization (Figure 3, Box 8). +## Reference documentation -![Workflow outline](docs/images/workflow.png) +- [Overview of the method and its limitations](docs/method-overview.md) +- [Description of the quality metrics reported by REViewer](docs/metrics.md) -This algorithm is based on the idea that if a given locus is sequenced well and each constituent -repeat is genotyped correctly, then it is possible to distribute the reads to achieve an even -coverage of each haplotype. (Although many reads may not be assigned to the correct haplotype of -origin, especially in cases when the repeats are homozygous, and the resulting haplotypes are -identical.) Conversely, if the size of a repeat is significantly overestimated or underestimated, -no assignment of reads will result in an even pileup making the genotyping error easy to notice. +## Companion tools -## Limitations +- [FlipBook](https://github.com/broadinstitute/flipbook) is an image server for +REViewer developed by [Ben Weisburd](https://github.com/bw2). It provides a +convenient way to inspect large quantities of read pileups. -REViewer is a tool for assessing consistency of sequencing data with repeat genotypes produced by -ExpansionHunter. It provides a mechanism for reviewing the evidence supporting a genotype call in -clinical settings and to identify problematic corner cases to drive future development. The read -pileup plots generated by REViewer may contain inaccuracies: The repeats may not be phased correctly -(e.g., when repeats are located far apart from each other) and read pairs consistent with both -haplotypes will often be assigned to the incorrect haplotype. Also, the current version of REViewer -visualizes repeats whose span does not exceed the fragment length and longer repeats are capped at -the fragment length. +- [Review BAMs](https://gitlab.com/andreassh/review-bams) is script that allows +applying REViewer to a regular BAM file (by running ExpansionHunter in the +background). It was developed by [Andreas Halman](https://gitlab.com/andreassh). -## External links +## Citation -- [A blog post describing the method](https://www.illumina.com/science/genomics-research/reviewer-visualizing-alignments-short-reads-long-repeat.html) +- Dolzhenko E, Weisburd B, Garikano K, Rajan Babu IS, and colleagues, +[REViewer: Haplotype-resolved visualization of read alignments in and around +tandem repeats](https://www.biorxiv.org/content/10.1101/2021.10.20.465046v1), +bioRxiv, 2021 diff --git a/docs/images/allele-depth-example.png b/docs/images/allele-depth-example.png new file mode 100644 index 0000000000000000000000000000000000000000..343306a4c82b7cf04448d4f00cc2e05062a6e261 GIT binary patch literal 20191 zcmb5W1yogExA1)s2>}5?x?5VhK|o4Mx*HA+(hVvg-AH$LcPc4x=#B%@-5ff;&HuUg zdGCAgxa0f2G0@E!?z8t=bIn+D&fhZRvx3xX6hagb2=rP;`lB)k1m6by{)qen_&Y%> zBL%p;`Xa661Onl`dHw@8NP8{<0+EAcK8mWkryW93HQ%Z|A)VAlx8xKdqZHwn1`{r4 zF&Uix$P0l&WcRF#L#9MH>cehJL@g6&7IX?OJVl4uxmYa;{vxoxD~JtC#0>1E`Hc5c 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a/docs/method-overview.md b/docs/method-overview.md new file mode 100644 index 000000000..b225e4892 --- /dev/null +++ b/docs/method-overview.md @@ -0,0 +1,43 @@ +# Overview of the method + +REViewer is designed to display alignments of reads generated by ExpansionHunter +(Figure 3, boxes 1-3). These alignments are obtained by realigning reads +originating in the target region to the corresponding sequence graph encoding one +or more repeats located there5. REViewer then constructs putative haplotype +sequences using genotypes produced by ExpansionHunter and then selects a pair of +haplotypes that have the highest consistency with the read alignments (Figure 3, +boxes 4-6). (This step is skipped for repeats on haploid chromosomes.) Next, +REViewer determines the set of possible alignment positions for each read pair +on each haplotype. For example, a read pair originating within a flanking +sequence shared by both haplotypes has exactly one alignment position on each +haplotype (Figure 3, Box 7a) while a read pair whose both mates are comprised +of the repeat sequence has multiple possible origins on haplotypes with +sufficiently long repeats (Figure 3, Box 7b). To generate a read pileup, +REViewer selects one alignment position at random for each read pair. This step +is repeated a specified number of times (10,000 by default) to generate multiple +pileups. The pileup with the most even coverage of each allele is selected for +visualization (Figure 3, Box 8). + +![Workflow outline](images/workflow.png) + +This algorithm is based on the idea that if a given locus is sequenced well and +each constituent repeat is genotyped correctly, then it is possible to +distribute the reads to achieve an even coverage of each haplotype. (Although +many reads may not be assigned to the correct haplotype of origin, especially in +cases when the repeats are homozygous, and the resulting haplotypes are +identical.) Conversely, if the size of a repeat is significantly overestimated +or underestimated, no assignment of reads will result in an even pileup making +the genotyping error easy to notice. + +## Limitations + +REViewer is a tool for assessing consistency of sequencing data with repeat +genotypes produced by ExpansionHunter. It provides a mechanism for reviewing the +evidence supporting a genotype call in clinical settings and to identify +problematic corner cases to drive future development. The read pileup plots +generated by REViewer may contain inaccuracies: The repeats may not be phased +correctly (e.g., when repeats are located far apart from each other) and read +pairs consistent with both haplotypes will often be assigned to the incorrect +haplotype. Also, the current version of REViewer visualizes repeats whose span +does not exceed the fragment length and longer repeats are capped at the fragment +length. diff --git a/docs/metrics.md b/docs/metrics.md new file mode 100644 index 000000000..2c8bed223 --- /dev/null +++ b/docs/metrics.md @@ -0,0 +1,26 @@ +# Quality metrics + +REViewer reports various summary measurements, called **quality metrics**, that +describe key properties of read pileups. Quality metrics make it possible to +automate assessment of large collections of STR genotype calls either (a) by +selecting a series of thresholds to stratify the value of each metric into +"good", "suspicious", and "bad" categories, or (b) by using more flexible +statistical / machine learning approaches. + +This document describes the quality metrics reported by REViewer. All quality +metrics are stored in a tab-separated (TSV) file `.metrics.tsv`. +If you have suggestions for additional metrics, please consider [creating an +issue](https://github.com/Illumina/REViewer/issues). + +## Allele depth + +In general, sequencing depth of a whole-genome sequencing sample is the average +number of reads that overlap any given genomic position in that sample. (For +example, a depth of 30x means that a base would be overlapped by 30 reads on +average.) The **allele depth** metric is an extension of this concept to STRs: +It reports the sequencing depth of each STR allele. The diagram below shows an +example of a well-genotyped repeat (left) where both STR alleles have the +expected sequencing depth and an example (right) where the size of the long +allele may be overestimated. + +![Example of allele depth metric](images/allele-depth-example.png)