[Faria]

We used the new kit but our bioinformatician has been unsuccessful with the demux using Nugen's protocol. We are trying to figure out the source of the problem.

[lyndaben]

If you figure it out, please would you post the solution? We are prepping libraries with the new kit at the moment, so it would be good to know before we have them sequenced

[Faria]

Will do! Please keep me posted on how yours turn out.

[luc]

Are the indices by chance not properly methylated or do they seem contaminated with unrelated sequences?

Demultiplexing rate with new kit is low with or without PhiX. Undetermined folder reads have index but it seems to have various sequences. NuGEN should have seen this issue if they sequenced libraries according to their recommendation.

[nucacidhunter]

Sequences of part of NuGEN P5 adapter is not in public domain (they use custom read 1 sequencing primer). I think they ligate partial adapters (methylated) and then add index and restore full adapter sequence after conversion with PCR. Undetermined reads’ index seems to be random indicating either sequencing or synthesis error. Sequencing error and sequencing primer synthesis issues can be excluded because other lanes in flow cell are demultiplexing with higher rate (95-99%). That narrows down the possible cause to adapter or PCR primer synthesis error.

They are not from contamination because reads have very low C and high T content indicating conversion and map to reference.

Edit: Further analysis indicates that they use Y adapters in which P5 sequences are non-methylated, but P7 sequences are.

[Faria]

We are now thinking that our demultiplexing issue is due to low diversity of the index pool. We had a pool of four libraries with the following indexes: GAGTCA, AGCATG, AAGAGG, and GGAGAA. According to their instructions, it shouldn't have been a problem but apparently, it is.

[nucacidhunter]

Those index sequences are color balanced. Illumina uses red laser for A/C and green laser for sequencing G/T residues. In each cycle at least one of each color should be present. Poor demultiplexing is seen even when all 16 index is multiplexed.

[Faria]

Well, there goes that theory!

[GenoMax]

@Faria: Do you know what the cluster density was for the run in question? We have found that the index sequencing is generally more sensitive to demultiplexing issues if the flowcell is overloaded or is very near so.

[Faria]

Cluster density was actually not high, just 886, 83% PF.

[nucacidhunter]

I had cluster densities intentially at 700s well below max. My explanation at post #20 seems most probable. They either have a faulty design or oligo synthesis issues. These issues should be resolved before a product launch or kit shipment. Their techsupport has been silent too.

[nucacidhunter]

I wonder if anyone have tried paired end sequencing of these libraries. Since index read is primed from similar region to R2 on P7 adapter, I would expect to see a drop in R2 quality or mapping rate as well.