First index always lowest percent reads

[creeves]

We usually multiplex 6 to 8 yeast genomes in one MiSeq run using 2x250bp paired end reads. So far we have always used N701 through N706 or N708 for the indices. Curiously, the percent reads for the N701 indexed genome is always the lowest, sometimes significantly, giving sub-optimal coverage of that genome. The percentage of all indexed reads is always about 98%. Any idea why N701 should give fewer reads than the other indices?

[kcchan]

How are you quantitating your libraries for sequencing? Are you using qPCR to verify and normalize each of your libraries or are you using Nextera XT where you're just using the bead normalization?

[creeves]

I use Picogreen to quantitate each library and a Bioanalyzer to get the average size of each. From this I normalize each library to 4 nM, pool, dilute to 2 nM to denature, and load 12 pM. This gives a cluster density around 1000 K/mm2 and a pretty even distribution of the reads, except that there are fewer N701 reads relative to the other indices. I have not found qPCR to be helpful in getting better results.

[thomasblomquist]

Bioanalyzer and interchelater dye is good for qualitative QC, but PCR is vastly superior to determine the actual number of copies that have appropriate adapter incorporation.

-Tom

[creeves]

Yes, qPCR is superior, but Picogreen plus Bioanalyzer is what Illumina recommends in their protocol. My original question remains unanswered, i.e. why should N701 give many fewer copies with appropriate adapter incorporation compared to the other indices?

[kcchan]

It could have something to do with that particular library or just a bad primer. Can you identify any unusual patterns with the sample using N701? Are the concentrations lower than the others or the Bioanalyzer peaks in a different position?

[creeves]

The lower number of reads assigned to N701 has correlated only with N701 regardless of the genomic DNA used to make that library. The lower number of reads has occurred in 2x150 bp and 2x250 bp runs. It has also occurred with different index primer kits and with different species of yeast. I am going to avoid N701 whenever possible.

[Chipper]

Have you looked at the undetermined reads if there are more reads with errors from N701?

[genomeseeker]

Is this across multiple library prep kit lot numbers? Maybe a marginal N701 adapter lot is in the kit you're using...

[creeves]

N701 still the worst index

The problem with N701 continues. The N701 library always has the lowest representation in a pool, often too low for good coverage. The % unassigned reads is low (less than 2%) and there is no way to determine if most are from the N701 index, because our pools consist of strains of yeast differing only by a few snps. Doesn't anyone have a good explanation for this?

[dkunz]

We recently had the same problem in our lab. According to Illumina the S501 adapter interferes with the N70X adapter. Unfortunately they didn't know why this happens.

[pardhu]

we generally go for Bioanalyzer to know the concentration and size distribution.. bioanalyzer based quantification is giving pretty good results..

[creeves]

universal i5 primer

Since I only need to use the i7 index reads to demultiplex ten yeast genomes per MiSeq run, the i5 primer does not need to have an index. I have a bunch of i5 primer minus an index, but wonder what concentration it should be (and what buffer it should be in) to be compatible with the Nextera PCR reaction. Does anyone out there know this?