Hi,
I'm doing metabarcoding of bat diet by amplifying a 157bp region of CO1.
I'm using illumina protocol for 16s metagenomic libraries, but with tag modified primers. This means that the PCR primers are composed of adaptor + 5bp tag + primer. I amplified 12 plates, using the same primer, but with different tags (1 different tag per plate). I then cleaned, quantified every plate, normalized and pooled the 12 plates into just 1. Afterwards I did the indexing PCR with illumina's nextera xt kit (96 combinations). This allowed the multiplexing of 12 x 96 samples.
However, I only got decent results of 6 plates (~5000 reads per sample), and the other 6 failed (~50 reads per sample).
Does anyone have any idea of why this might happen? Could my tags affect the ability of miseq reading the dna?
Thanks in advance!
Vanessa
ps: for some reason my cluster density was also low (400), although the library was at 4nm (quantified using qpcr and tapestation) and diluted to 12pm.
I'm doing metabarcoding of bat diet by amplifying a 157bp region of CO1.
I'm using illumina protocol for 16s metagenomic libraries, but with tag modified primers. This means that the PCR primers are composed of adaptor + 5bp tag + primer. I amplified 12 plates, using the same primer, but with different tags (1 different tag per plate). I then cleaned, quantified every plate, normalized and pooled the 12 plates into just 1. Afterwards I did the indexing PCR with illumina's nextera xt kit (96 combinations). This allowed the multiplexing of 12 x 96 samples.
However, I only got decent results of 6 plates (~5000 reads per sample), and the other 6 failed (~50 reads per sample).
Does anyone have any idea of why this might happen? Could my tags affect the ability of miseq reading the dna?
Thanks in advance!
Vanessa
ps: for some reason my cluster density was also low (400), although the library was at 4nm (quantified using qpcr and tapestation) and diluted to 12pm.
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