Miseq Genomic DNA analysis
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6.9 years ago

We have sequenced genomic dna using miseq 2x250, and have received fastq files, i was wondering how to go about with respect to alignment of these reads and check for indels and snps is there a specific work flow?

genomic dna alignment • 1.6k views
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which species are you sequencing?

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we are working on mouse

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6.9 years ago

Well, you could use the BBMap package and do something like this:

#Remove duplicates
clumpify.sh in=reads.fq.gz out=clumped.fq.gz dedupe optical

#Remove low-quality regions
filterbytile.sh in=clumped.fq.gz out=filtered_by_tile.fq.gz

#Trim adapters
bbduk.sh in=filtered_by_tile.fq.gz out=trimmed.fq.gz ktrim=r k=23 mink=11 hdist=1 tbo tpe minlen=100 ref=bbmap/resources/adapters.fa ftm=5 ordered

#Remove synthetic artifacts and spike-ins.
bbduk.sh in=trimmed.fq.gz out=filtered.fq.gz k=27 ref=bbmap/resources/sequencing_artifacts.fa.gz,bbmap/resources/phix174_ill.ref.fa.gz ordered qrtim=r trimq=6

#Map to reference
bbmap.sh in=filtered.fq.gz out=mapped.sam.gz bs=bs.sh pigz unpigz ref=reference.fa

#Call variants
callvariants.sh in=mapped.sam.gz out=vars.txt vcf=vars.vcf.gz ref=reference.fa ploidy=1 prefilter
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For WGS MiSeq runs first two steps should not be needed.

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I'll have to look into that. I don't see much MiSeq 2x250bp WGS data, mainly 2x300 amplicon data, which gets pretty ragged toward the ends. I have noticed, though, that MiSeq appears to have a pretty consistent positional quality component (at least in the sample I analyzed for that purpose) that appears to be due to focus (the number of mismatches increased radially out from the center).

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Thank you,I will ask my admin to install bbMap meanwhile can this also be done using bwa mem?

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