Hi there!
We have a big problem with our patients bam files. We are using IGV software to visualize bam files from our sequencing platform which is new Illumina Miniseq.
Everything seems to be normal but when we look at IGV we see something which is more diffrent than we expected to see.
I expected to see some alternative reads, variable coverage in some exons, but in our Miniseq bam files we see something like this:
https://postimg.org/image/b2glwh9ad/
Some reads are just green or red... I expected to see a single nucleotide change in shades of green for example.
In my experience I've analyzed bam files where many same reads was a signal of machines mistake during mapping or sequencing. In this photo every read is the same.
In this photo you can also see that there are some gaps and some exons seem to be not mapped.
I have never seen such thing before so meybe someone have the same problem or already tried Illumina Miniseq platform?
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Which kit/protocol did you use to prepare the library?
It's a standard Illumina procedure for Miniseq library preparation from their workflow guides.
Have never used the MiniSeq - but it looks like loads of illumina kits are available: http://www.illumina.com/systems/miniseq/kits.html
Which one did you use?
I think it was TruSeq Custom Amplicon v1.5
Not sure what you mean by this, but it appear to be amplicons and they should have the same start position and read length (at least on Illumina).
I've upload to IGV 3 patiens now. The first and the second one are from bam files which were generated by Miniseq. The last one is from another run which was made on Miseq some months ago. I'm realy not sure why they are so diffrent. You can see that covarage on the last one is described by "mountainlike" graph. In Miniseq there are squares.
https://postimg.org/image/9r8ebm3xt/
So my question is: Are those bam files correct or maybe something went wrong on the stage of Miniseq analysis?
My guess is that 2 different methods were used to create a library, the new one you sequenced on the miniseq could be a TruSeq library, but the older one looks more like a shotgun. And in a shotgun you would expect something 'mountainlike'.
If that is true, the BAM files are correct.
I can't come up with any explanation as to why using the miniseq would make squares out of mountains.