Average Insert Size

[hamcan]

Hi there,
I ran an Illumina HiSeq run 2x250 and wanted to know how I could find out the average insert size from my fastq files?

Thanks in advance!

[Brian Bushnell]

There are three primary ways. I'll describe how to do them using the BBMap package.

1) Via mapping, which requires a reference:
bbmap.sh in1=r1.fastq in2=r2.fastq ref=ref.fasta ihist=ihist.txt reads=2m pairlen=2000

2) Via overlap, which requires overlapping reads (they probably overlap given you ran at 2x250):
bbmerge.sh in1=r1.fastq in2=r2.fastq ihist=ihist.txt reads=2m

3) Via assembly, which requires sufficient read depth and memory to assemble the genome:
bbmerge-auto.sh in1=r1.fastq in2=r2.fastq ihist=ihist.txt extend2=200

2 is the fastest. The best choice and best settings depend on your data, though. Can you describe the organism, experiment, and target insert size?

[hamcan]

Thanks for responding so quickly!
Well I ran a HiSeq run on environmental water samples. So it's a metagenomics experiment. The purpose of the experiment is to look at what species are predominant in certain bodies of water. I am looking at bacterial species more specifically.

I am going to run trimmomatic to trim my reads and remove adapter sequences. Afterwards I am going to use FLASh to merge my reads. In order to find the correct parameters to use for FLASh I need to figure out my average insert size. Hence that is why I am trying to find out how to do that.

[Brian Bushnell]

Full disclosure - I developed BBDuk and BBMerge. In my testing, BBDuk has greater accuracy than Trimmomatic, and BBMerge has greater accuracy than Flash. But you can certainly determine your insert size with BBMerge and then use that with Flash, if you wish. In my experience that's not necessary; Flash will not give substantially better output even when you know the average insert size apriori. Rather, it will output a helpful message when you give it settings it finds inappropriate. You can then correct them, but it will still yield similar results, in my experience.

Unless you answer all of the questions posed, nobody can give you optimal advice... for example, what's the target insert size?