I'm working on a project where I am analyzing the performance of an alignment workflow. My goal is to find regions in the resulting BAM file where there are outstanding discrepancies or anything that indicates my assembly/alignment has "mistakes". I'm working with human genomes from the 1000 genomes project, they are high coverage Illumina paired end reads, each read is 100bp.
My workflow so far:
- input paired end FASTQ files (human) into SPAdes to create contigs.fasta file
- align paired end FASTQ files to contigs.fasta file with minimap2 to get SAM file
- convert SAM to BAM with samtools
- sort and index BAM file
- load BAM file into IGV and load contigs.fasta as the reference
In the IGV GUI, I have colored alignments by insert size and pair orientation. This has revealed a bunch of insertions, tandem duplications, and inversions.
I am wondering:
- Do these colored reads indicate that the assembler made "mistakes" or could they be there just by chance?
- Do SNPs indicate anything about how SPAdes/minimap2/samtools performed?
- Is there a better way to sort/color the alignments based on what I'm trying to do?
Thank you so much to anyone who has any advice!
EDIT: here is a screenshot of the alignment. For clarification, there are 8 genomes of interest, hence 8 tracks
Background Integrative genome viewer (IGV) is a well known Broad Institute tool and available here.
The most recent papers are: James T. Robinson, Helga Thorvaldsdóttir, Aaron M. Wenger, Ahmet Zehir, Jill P. Mesirov; Variant Review with the Integrative Genomics Viewer. Cancer Res 1 November 2017; 77 (21): e31–e34. https://doi.org/10.1158/0008-5472.CAN-17-0337