Working with Alignment Data

This tutorial details how to use the OpenCGA alignment command line to run the alignment/mapping pipeline steps. The alignment pipeline outputs alignments in BAM files from raw sequence data in FastQ format files. BAM files can be used for further analysis, such as alignment statistics, coverage computation or variant calling.

Prerequisites 

A working setup of OpenCGA is required to setup a testing environment, please follow the steps on installation guide.

In addition, you need to download the following data files:

• Raw sequence data file: input.fastq
• Reference genome: reference.fasta

The alignment pipeline

Quality control for raw sequence data: FastQC subcommand

In order to use the input.fastq file, it has to be linked to the OpenCGA catalog:

$ ./opencga.sh files link -i ~/input.fastq --path test/ --parents

Once linked the FastQ file, you can run the FastQC command:

$ ./opencga.sh alignments fastqc --file input.fastq

For the input.fastq file, the FastQC command creates a report file called input_fastqc.html that can be downloaded from the OpenCGA catalog to the local directory /tmp by using the following command:

$ ./opencga.sh files download --file input_fastqc.html --to /tmp

Here is the FastQC report file: input_fastqc.html.

Mapping raw sequences: BWA subcommand

First, link the reference.fasta file to the OpenCGA catalog:

$ ./opencga.sh files link -i ~/reference.fasta --path test/ --parents

Then, you can run the bwa index command to index database sequences in the FASTA format:

$ ./opencga.sh alignments bwa --command index --fasta-file reference.fata

The index for the reference.fasta file created by the bwa index command consists of the following files:

reference.fasta.bwt
reference.fasta.pac
reference.fasta.ann
reference.fasta.amb
reference.fasta.sa

Once created the index, you can map the FastQ file by using the bwa mem command:

$ ./opencga.sh alignments bwa --command mem --index-base-file reference.fasta --fastq1-file input.fastq --sam-file output.sam

In the previous command, the result alignments are saved in SAM format in the output.sam file .

Converting to and sorting BAM files

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Indexing and querying BAM files

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Computing and querying BAM coverage

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Computing and querying BAM statistics

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