Total reads from MiSeq 16S V4 PE run

[Egansbay]

Hi everyone

Probably a stupid question, but i'm finding it hard to find meaningful info on sequencing depth and read numbers on the MiSeq.

Illumina states that a 16S V4 run should deliver ~20m reads.

Assuming 96 samples are run, that's 208,333 raw reads per sample.

Therefore, if I run 24 samples, should I expect 833,333 raw reads per sample?

I guess my question is why does the number of sequences fall with increased samples?

Thank you

[GW_OK]

An Illumina sequencer will, or at least should, generate the same amount of reads for every run, respective to the chemistry loaded on it. A v3 Miseq kit should give about ~20M reads per run.

Much like a pizza, every time you split it it the slices become smaller.

Slicing a pizza (evenly) 24 times will give you larger pieces over slicing it 96 times.

[Egansbay]

Thank you for your reply GW_OK

So from the number of ~20m reads, Illumina states that you should get ~100,000 seqs per sample, assuming 96 samples were run.

Do you think that this number refers to 100,000 OTU classified seqs after quality filtering, given that the raw reads from 96 samples should be >200,000 per sample?

Thank you again

[GW_OK]

I don't know the context of Illumina's statement, so I can't comment.

[Egansbay]

I am just referring to the statement in the MiSeq wet lab protocol where it reads "The MiSeq run output is >20m reads, and assuming 96 indexed samples, can generate >100,000 reads per sample, commonly recognized as sufficient for metagenomic surveys".

http://www.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf

[GW_OK]

Sounds about right, then.

I wouldn't run 2x300's on the Miseq, though, if you're planning on doing that.

[Egansbay]

Thank you - I have run 2 x 300bp, but have to trim as the quality of the reverse read is not that good. Assume this is what you're referring to

[thermophile]

20M is if you are loading the max which you really shouldn't for amplicons. I run v2 2x250, aiming for 700-800k clusters/mm, ~80% passing filter, so ~8-10M reads which includes the phiX (I'm running 15%). Also remember that your sequences will not be perfectly distributed between the samples.

[Egansbay]

Thanks Thermophile - I think the seq co must have loaded the max, as 24 samples yielded 22m raw reads.

Not sure if the seq was too deep though, as when I run rarefaction down to 100,000 reads/sample it still looks ok