(howto) Map and mark duplicates
Posted in Tutorials | Last updated on 2014-02-26 23:24:55


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Objective

Map the read data to the reference and mark duplicates.

Prerequisites

  • TBD

Steps

  1. Identify read group information
  2. Generate a SAM file containing aligned reads
  3. Convert to BAM, sort and mark duplicates

1. Identify read group information

The read group information is key for downstream GATK functionality. The GATK will not work without a read group tag. Make sure to enter as much metadata as you know about your data in the read group fields provided. For more information about all the possible fields in the @RG tag, take a look at the SAM specification.

Action

Compose the read group identifier in the following format:

@RG\tID:group1\tSM:sample1\tPL:illumina\tLB:lib1\tPU:unit1 

where the \t stands for the tab character.


2. Generate a SAM file containing aligned reads

Action

Run the following BWA command:

In this command, replace read group info by the read group identifier composed in the previous step.

bwa mem -M -R ’<read group info>’ -p reference.fa raw_reads.fq > aligned_reads.sam 

replacing the <read group info> bit with the read group identifier you composed at the previous step.

The -M flag causes BWA to mark shorter split hits as secondary (essential for Picard compatibility).

Expected Result

This creates a file called aligned_reads.sam containing the aligned reads from all input files, combined, annotated and aligned to the same reference.

Note that here we are using a command that is specific for pair ended data in an interleaved fastq file, which is what we are providing to you as a tutorial file. To map other types of datasets (e.g. single-ended or pair-ended in forward/reverse read files) you will need to adapt the command accordingly. Please see the BWA documentation for exact usage and more options for these commands.


3. Convert to BAM, sort and mark duplicates

These initial pre-processing operations format the data to suit the requirements of the GATK tools.

Action

Run the following Picard command to sort the SAM file and convert it to BAM:

java -jar SortSam.jar \ 
    INPUT=aligned_reads.sam \ 
    OUTPUT=sorted_reads.bam \ 
    SORT_ORDER=coordinate 

Expected Results

This creates a file called sorted_reads.bam containing the aligned reads sorted by coordinate.

Action

Run the following Picard command to mark duplicates:

java -jar MarkDuplicates.jar \ 
    INPUT=sorted_reads.bam \ 
    OUTPUT=dedup_reads.bam \
    METRICS_FILE=metrics.txt

Expected Result

This creates a sorted BAM file called dedup_reads.bam with the same content as the input file, except that any duplicate reads are marked as such. It also produces a metrics file called metrics.txt containing (can you guess?) metrics.

Action

Run the following Picard command to index the BAM file:

java -jar BuildBamIndex.jar \ 
    INPUT=dedup_reads.bam 

Expected Result

This creates an index file for the BAM file called dedup_reads.bai.


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