2. Alignment

Overview

After the barcodes have been identified, the reads need to be aligned to the reference genome. In our standard ligation scheme, only read 1 contains genomic DNA, so only read 1 is aligned. We do not perform a paired-end alignment, despite having paired-end reads.

Usage

Any aligner will work. We use bowtie2 to illustrate.

bowtie2 --local --trim5 11 -x index -U example.R1.barcoded.fastq.gz -S example.sam

At the start of each read is a DPM sequence which doesn't correspond to any genomic region. We trim off the first eleven bases of each read to avoid aligning the DPM sequence. In addition, it is possible for read 1 to extend through the genomic DNA sequence into the tags on the other end of the fragment. We perform a local alignment, rather than a global alignment to account for this possibility.

Input and output files

Example input:

@HISEQ:623:HY5KHBCXX:2:2206:7231:7108::[DPM6A5][NYBot35_Stg][NOT_FOUND][Even2Bo19][Odd2Bo69]
ATTGGTAGGTCGGAATTGCACGCTGTAGCGGCATGCTGATGGAGAGGAGAGACTTCTAGCTAGCTACGTGACTGATCCGCACACTGCGACACGTGATCGC
+
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII

Example output:

HISEQ:623:HY5KHBCXX:2:2206:7231:7108::[DPM6A5][NYBot35_Stg][NOT_FOUND][Even2Bo19][Odd2Bo69]	0	chr1	17644	255	100M	*	0	0	ATTGGTAGGTCGGAATTGCACGCTGTAGCGGCATGCTGATGGAGAGGAGAGACTTCTAGCTAGCTACGTGACTGATCCGCACACTGCGACACGTGATCGC
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII		NM:i:0	MD:Z:100