Picard

Overview

Picard is a set of Java command-line tools maintained by the Broad Institute for manipulating high-throughput sequencing data. It is best known for marking PCR duplicates and collecting a wide range of alignment quality metrics. Picard integrates tightly with GATK-based variant-calling pipelines and produces detailed metrics files that can be visualised with tools such as MultiQC.

Installation

mamba install -c bioconda picard

Basic Usage

Mark PCR duplicates

java -jar picard.jar MarkDuplicates \
  I=sample.sorted.bam \
  O=sample.dedup.bam \
  M=sample.dup_metrics.txt \
  REMOVE_DUPLICATES=false

Collect alignment summary metrics

java -jar picard.jar CollectAlignmentSummaryMetrics \
  R=reference.fa I=sample.dedup.bam O=alignment_metrics.txt

Collect insert size distribution

java -jar picard.jar CollectInsertSizeMetrics \
  I=sample.dedup.bam O=insert_metrics.txt H=insert_hist.pdf

Key Parameters

Flag / option	Description
`I`	Input BAM or SAM file.
`O`	Output BAM or metrics file.
`M`	Duplication metrics output file (used with `MarkDuplicates`).
`R`	Reference FASTA file (required by several collectors).
`REMOVE_DUPLICATES`	If `true`, discard duplicate reads from the output BAM instead of only flagging them (default: `false`).
`H`	Histogram PDF output file (used with `CollectInsertSizeMetrics`).
`VALIDATION_STRINGENCY`	How strictly to validate the input (`STRICT`, `LENIENT`, or `SILENT`). Set to `LENIENT` or `SILENT` to skip non-critical format warnings.
`CREATE_INDEX`	If `true`, create a BAM index file alongside the output BAM (default: `false`).

Expected Output

MarkDuplicates – a BAM file with duplicate FLAG bits set (or removed) and a tab-delimited metrics file reporting the number and fraction of duplicates per library.
CollectAlignmentSummaryMetrics – a text file with statistics for each read category (paired, unpaired, first-of-pair, second-of-pair) including total reads, aligned reads, mismatch rate, and more.
CollectInsertSizeMetrics – a text metrics file and an accompanying PDF histogram showing the distribution of insert sizes for paired-end libraries.