[groad]

I don't think balanced barcodes will work. The index read occurs after read 1 so the instrument will still have issues calling clusters.

I am doing some tag sequencing that requires a custom library prep protocol, and I have the same issue with homogeneous nucleotides for the first 10 bases of read 1. Illumina tech support recommended adding a stretch of 12 Ns (not 6) right after the read 1 primer binding site. It seems like that would be fairly straightforward to add to primers for amplicon sequencing.

I am pretty sure (but don't quote me on this) that you can specify indeces of different lengths on the sample sheet. I think that the 2 index reads use a 6 base and a 7 base index.

Hi bbeitzel,

Do you mean to add 12 degenerated nucleotides to the 5' end of gene specific primer? Thanks.

Jim

[ramsemm]

Hi!

I am doing some similar sequencing - 30 samples of 1 amplified region. I was wondering if anyone has a citation or comment on what level is the limit for assigning taxonomy to 100 bp sequences from bacterial 16S (V4) amplicons?

Thank you in advance,
Molli

[bbeitzel]

groad,

If you have incorporated the Illumina-specific sequences (sequence read primer binding sites, bridge PCR sequences, index read primer site) into your primers, then the 12 Ns would come between the Illumina stuff and the gene-specific sequence. If you are adding the Illumina adapters after PCR (ie. running amplicons through the TruSeq protocol), then you would put the 12 Ns at the 5' ends of both PCR primers. They have to be on both primers since TruSeq will not distinguish one end from the other.

Hope this helps.

[vl80]

Apologies for reactivating an old thread. I posted a similar question yesterday and thought may be asking here may get more answers.
I plan to sequence fragments ranging from 90 bp - 450 bp using Miseq as a pilot study. Per sample, I would have 2 amplicons. My PCR is a multiplex with > 10 primer pairs per reaction. I am completely new to this field and my theorising was to use the Trueseq kit, skip the fragmentation and end repair steps and proceed with the other steps. However, reading this thread may me doubt the appropriateness of that method. I am worried about suggetion to modify the PCR primers with an N sequence as this may cause mispriming in my multiplex PCR. Any suggestions are welcome. Thanks a lot

[pmiguel]

Modulo any special issues with v2 hardware, the "spike in 50% phiX" seems to be a cure-all. Using v1 hardware we did a v3 loop sequence where the first 20 bases were 100% identical. But the addition of a genomic DNA library allowed this to work fine. So just use that method initially and you should be fine.

--
Phillip

[LejzerowiczF]

for diversity analysis of communities, using long primer constructs
such as [P5][Seq Primer]NNNNN[Target Primer]
revives the long-known primer bias (see Berry et al. 2011 http://aem.asm.org/content/77/21/7846.short)
its best to design short, anti-complementary tags (the shortest the easiest to satisfy anti-complemetarity).

short primers + combinatorics + 1 lib prep => cheap solution

moreover, adding tags in amplicons make their first, hypervariable bases tobe sequenced in the first miseq cycles... => high signal entropy => good sequencing quality.

to be acounted: short tags will be in the sequences so you only loose few postions over the read length...

for all recommendation + how to avoid tags swiching cross-contamionations resulting (mostly) from the lib- prep: see Esling et al. 2015 http://nar.oxfordjournals.org/conten...ar.gkv107.full

good luck with your data!

F