Collects a suite of metrics to QC duplex sequencing data. Inputs ------ The input to this tool must be a BAM file that is either: 1. The exact BAM output by the 'GroupReadsByUmi' tool (in the sort-order it was produced in) 2. A BAM file that has MI tags present on all reads (usually set by 'GroupReadsByUmi' and has been sorted with 'SortBam' into 'TemplateCoordinate' order.

Calculation of metrics may be restricted to a set of regions using the '--intervals' parameter. This can significantly affect results as off-target reads in duplex sequencing experiments often have very different properties than on-target reads due to the lack of enrichment. Several metrics are calculated related to the fraction of tag families that have duplex coverage. The definition of \"duplex\" is controlled by the '--min-ab-reads' and '--min-ba-reads' parameters. The default is to treat any tag family with at least one observation of each strand as a duplex, but this could be made more stringent, e.g. by setting '--min-ab-reads=3 --min-ba-reads=3'. If different thresholds are used then '--min-ab-reads' must be the higher value. Outputs ------- The following output files are produced: 1. <output>.family_sizes.txt: metrics on the frequency of different types of families of different sizes 2. <output>.duplex_family_sizes.txt: metrics on the frequency of duplex tag families by the number of observations from each strand 3. <output>.duplex_yield_metrics.txt: summary QC metrics produced using 5%, 10%, 15%...100% of the data 4. <output>.umi_counts.txt: metrics on the frequency of observations of UMIs within reads and tag families 5. <output>.duplex_qc.pdf: a series of plots generated from the preceding metrics files for visualization 6. <output>.duplex_umi_counts.txt: (optional) metrics on the frequency of observations of duplex UMIs within reads and tag families. This file is only produced if the '--duplex-umi-counts' option is used as it requires significantly more memory to track all pairs of UMIs seen when a large number of UMI sequences are present.

Within the metrics files the prefixes 'CS', 'SS' and 'DS' are used to mean: * CS: tag families where membership is defined solely on matching genome coordinates and strand * SS: single-stranded tag families where membership is defined by genome coordinates, strand and UMI; ie. 50/A and 50/B are considered different tag families. * DS: double-stranded tag families where membership is collapsed across single-stranded tag families from the same double-stranded source molecule; i.e. 50/A and 50/B become one family

Requirements ------------ For plots to be generated R must be installed and the ggplot2 package installed with suggested dependencies. Successfully executing the following in R will ensure a working installation: install.packages(\"ggplot2\", repos=\"http://cran.us.r-project.org\", dependencies=TRUE)

Collects a suite of metrics to QC duplex sequencing data. Inputs ------ The input to this tool must be a BAM file that is either: 1. The exact BAM output by the 'GroupReadsByUmi' tool (in the sort-order it was produced in) 2. A BAM file that has MI tags present on all reads (usually set by 'GroupReadsByUmi' and has been sorted with 'SortBam' into 'TemplateCoordinate' order.

Calculation of metrics may be restricted to a set of regions using the '--intervals' parameter. This can significantly affect results as off-target reads in duplex sequencing experiments often have very different properties than on-target reads due to the lack of enrichment. Several metrics are calculated related to the fraction of tag families that have duplex coverage. The definition of \"duplex\" is controlled by the '--min-ab-reads' and '--min-ba-reads' parameters. The default is to treat any tag family with at least one observation of each strand as a duplex, but this could be made more stringent, e.g. by setting '--min-ab-reads=3 --min-ba-reads=3'. If different thresholds are used then '--min-ab-reads' must be the higher value. Outputs ------- The following output files are produced: 1. <output>.family_sizes.txt: metrics on the frequency of different types of families of different sizes 2. <output>.duplex_family_sizes.txt: metrics on the frequency of duplex tag families by the number of observations from each strand 3. <output>.duplex_yield_metrics.txt: summary QC metrics produced using 5%, 10%, 15%...100% of the data 4. <output>.umi_counts.txt: metrics on the frequency of observations of UMIs within reads and tag families 5. <output>.duplex_qc.pdf: a series of plots generated from the preceding metrics files for visualization 6. <output>.duplex_umi_counts.txt: (optional) metrics on the frequency of observations of duplex UMIs within reads and tag families. This file is only produced if the '--duplex-umi-counts' option is used as it requires significantly more memory to track all pairs of UMIs seen when a large number of UMI sequences are present.

Within the metrics files the prefixes 'CS', 'SS' and 'DS' are used to mean: * CS: tag families where membership is defined solely on matching genome coordinates and strand * SS: single-stranded tag families where membership is defined by genome coordinates, strand and UMI; ie. 50/A and 50/B are considered different tag families. * DS: double-stranded tag families where membership is collapsed across single-stranded tag families from the same double-stranded source molecule; i.e. 50/A and 50/B become one family

Requirements ------------ For plots to be generated R must be installed and the ggplot2 package installed with suggested dependencies. Successfully executing the following in R will ensure a working installation: install.packages(\"ggplot2\", repos=\"http://cran.us.r-project.org\", dependencies=TRUE)