RNA-seq Bioinformatics

Introduction to bioinformatics for RNA sequence analysis

Alignment Visualization


RNA-seq_Flowchart3


Before we can view our alignments in the IGV browser we need to index our BAM files. We will use samtools index for this purpose. For convenience later, index all bam files.

    echo $RNA_ALIGN_DIR
    cd $RNA_ALIGN_DIR
    find *.bam -exec echo samtools index {} \; | sh

Visualize alignments

Start IGV on your laptop. Load the UHR.bam & HBR.bam files in IGV. You can load the necessary files in IGV directly from your web accessible amazon workspace (see below) using ‘File’ -> ‘Load from URL’. You may wish to customize the track names as you load them in to keep them straight. Do this by right-clicking on the alignment track and choosing ‘Rename Track’.

UHR hisat2 alignment:

http://YOUR_DNS_NAME/workspace/rnaseq/alignments/hisat2/UHR.bam

HBR hisat2 alignment:

http://YOUR_DNS_NAME/workspace/rnaseq/alignments/hisat2/HBR.bam

Go to an example gene locus on chr22:

Exercise

Try to find a variant position in the RNAseq data:

BAM Read Counting

Using one of the variant positions identified above, count the number of supporting reference and variant reads. First, use samtools mpileup to visualize a region of alignment with a variant.

    cd $RNA_HOME
    mkdir bam_readcount
    cd bam_readcount

Create faidx indexed reference sequence file for use with mpileup

    echo $RNA_REF_FASTA
    samtools faidx $RNA_REF_FASTA

Run samtools mpileup on a region of interest

    samtools mpileup -f $RNA_REF_FASTA -r 22:18918457-18918467 $RNA_ALIGN_DIR/UHR.bam $RNA_ALIGN_DIR/HBR.bam

Each line consists of chromosome, 1-based coordinate, reference base, the number of reads covering the site, read bases and base qualities. At the read base column, a dot stands for a match to the reference base on the forward strand, a comma for a match on the reverse strand, ACGTN for a mismatch on the forward strand and acgtn for a mismatch on the reverse strand. A pattern \+[0-9]+[ACGTNacgtn]+ indicates there is an insertion between this reference position and the next reference position. The length of the insertion is given by the integer in the pattern, followed by the inserted sequence. See samtools pileup/mpileup documentation for more explanation of the output:

Now, use bam-readcount to count reference and variant bases at a specific position. First, create a bed file with some positions of interest (we will create a file called snvs.bed using the echo command).

It will contain a single line specifying a variant position on chr22 e.g.:

22 38483683 38483683

Create the bed file

    echo "22 38483683 38483683"
    echo "22 38483683 38483683" > snvs.bed

Run bam-readcount on this list for the tumor and normal merged bam files

    bam-readcount -l snvs.bed -f $RNA_REF_FASTA $RNA_ALIGN_DIR/UHR.bam 2>/dev/null
    bam-readcount -l snvs.bed -f $RNA_REF_FASTA $RNA_ALIGN_DIR/HBR.bam 2>/dev/null

Now, run it again, but ignore stderr and redirect stdout to a file:

    bam-readcount -l snvs.bed -f $RNA_REF_FASTA $RNA_ALIGN_DIR/UHR.bam 2>/dev/null 1>UHR_bam-readcounts.txt
    bam-readcount -l snvs.bed -f $RNA_REF_FASTA $RNA_ALIGN_DIR/HBR.bam 2>/dev/null 1>HBR_bam-readcounts.txt

From this output you could parse the read counts for each base

    cat UHR_bam-readcounts.txt | perl -ne '@data=split("\t", $_); @Adata=split(":", $data[5]); @Cdata=split(":", $data[6]); @Gdata=split(":", $data[7]); @Tdata=split(":", $data[8]); print "UHR Counts\t$data[0]\t$data[1]\tA: $Adata[1]\tC: $Cdata[1]\tT: $Tdata[1]\tG: $Gdata[1]\n";'
    cat HBR_bam-readcounts.txt | perl -ne '@data=split("\t", $_); @Adata=split(":", $data[5]); @Cdata=split(":", $data[6]); @Gdata=split(":", $data[7]); @Tdata=split(":", $data[8]); print "HBR Counts\t$data[0]\t$data[1]\tA: $Adata[1]\tC: $Cdata[1]\tT: $Tdata[1]\tG: $Gdata[1]\n";'

PRACTICAL EXERCISE 7

Assignment: Index your bam files from Practical Exercise 6 and visualize in IGV.

Questions

Solution: When you are ready you can check your approach against the Solutions.