Hi everyone,
First post here, sorry for the bad writing.
Me and my colleagues are planning to do a Miseq run to examine the 16S RNA of several bacterial samples.
We would like to sequence as many samples as possible in one run, and we are thinking to use the 96 index nextera kit coupled with de v2 chemistry to sequence the V3-V4 region.
Our concerns are about 4 points:
1-Combining 2x250 PE and V3-V4 (460bp) works allright?
2-How much PhiX we should use to avoid overcluster? We were told that 30% should be enough.
3-As the number of samples is quite large, we are not sure if we’ll be able to quantify all samples through qPCR. Does a QUBIT based normalization of individual amplicons and a qPCR based normalization of pooled amplicons work? How badly could this bias our run?
4-If someone usually does this run, how many reads per sample are expected to be yielded?
Any help would be appreciated.
Cheers
First post here, sorry for the bad writing.
Me and my colleagues are planning to do a Miseq run to examine the 16S RNA of several bacterial samples.
We would like to sequence as many samples as possible in one run, and we are thinking to use the 96 index nextera kit coupled with de v2 chemistry to sequence the V3-V4 region.
Our concerns are about 4 points:
1-Combining 2x250 PE and V3-V4 (460bp) works allright?
2-How much PhiX we should use to avoid overcluster? We were told that 30% should be enough.
3-As the number of samples is quite large, we are not sure if we’ll be able to quantify all samples through qPCR. Does a QUBIT based normalization of individual amplicons and a qPCR based normalization of pooled amplicons work? How badly could this bias our run?
4-If someone usually does this run, how many reads per sample are expected to be yielded?
Any help would be appreciated.
Cheers
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