Gray bar shape, soft-clipped bases, backwards reference
hur...@georgetown.edu
Hello: I am new to NGS and IGV and have some questions that I couldn’t answer with the help manual. Can someone point me in the right direction? I am using Illumina Miseq and Basespace to analyze paired reads of Nextera library created from PCR amplification of my gene of interest. I have created an alignment in Basespace, trimmed off adaptors, and am now looking at the bam files in IGV.
1. Each sequence read is represented by a gray bar with variant positions marked. I understand that the arrow at the end of the gray bar indicates the direction of the read but when I view the sequences in paired view, I also see gray bars without arrows (blunt ended) and gray bars with arrows at both ends. I know that the shorter fragments might be completely sequenced in both directors—how will they appear in terms of the gray bar shape? What do these different gray bars mean?
2. I have not heard the term “soft-clipped bases” before.
I see that I have regions of sequence that are “soft-clipped”. What does
this mean? I noticed the presence of low frequency sequences that don’t match
my alignment and these appear to have been created by “soft-clipping”.
3. I am comparing my sequence to hg19, the human reference sequence. Two of the genes in this reference (chromosome 6, HLA-B, HLA-C) are “backwards”. This means that the coding sequence runs from right to left and the sequence reads themselves are the complement of the coding sequence. I see the little arrow at bottom right where I can switch strands for the reference sequence but is there a way in IGV to flip around my reads and the gene so I am reading 5’ to 3’ and reading the variation in the coding sequence?
4. When I access IGV through Basespace, I am asked to select a “project” before IGV will launch. Since my Basespace project files are fastq.gz files, there aren’t any bam or vcf files in the project file to work on in IGV. I have solved this by just opening bam files from my run so this is just a fyi for you.
5. I noticed that the IGV preferences should be set to show all reads, not set on “downsize reads”, in order to see the “view as pairs” correctly. I noticed that the manual didn’t explain this so this is just a fyi.
Thanks for the advice! Carolyn
Jacob Silterra
Basespace has their own version of IGV, you'll need to contact them for support on most of these issues.
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Jacob Silterra
Software Engineer
Broad Institute
Carolyn Hurley
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Carolyn Hurley
Each sequence read is represented by a gray bar with variant positions marked. I understand that the arrow at the end of the gray bar indicates the direction of the read but when I view the sequences in paired view, I also see gray bars without arrows (blunt ended) and gray bars with arrows at both ends. What do these different gray bars mean?
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