calculating expected reads

[Seqasaurus]

Hi,

we are currently trying to decide how to process our 16S amplicons on titanium. How would I come to a rough estimate of expected reads per sample on 1/8th PTP at 16-plex? (assuming everything is perfectly normalized).

I am under the impression that splitting the plate reduces sequencing output overall but am unsure as to how great a decrease to expect in output to expect.

Cheers,
Paul

[ulz_peter]

Hi,

The sequencing method manual states that with the 1/8th PTP you could assume 80,000-120,000 reads. That means 5000-7500 reads per sample at 16-plex assuming perfect normalization of the samples.
Furthermore it says one could expext 450,000-650,000 reads per region when dividing the plate in 2 parts which would result in 900,000-1,300,000 reads per plate. Dividing the plate in 8 parts could yield a maximum of 640,000 - 960,000 reads / plate.

Best regards,
Peter

[RCJK]

Hi there,

The number of reads output for amplicons is less than for shotgun libraries. 1/8th of a PTP should give you 65,000-100,000 HQ reads. A half plate would give 360,000-520,000. These numbers are from Tech Bulletin 13-2009 re: Amplicon Fusion Primer design guidelines.