[HESmith]

random (adj.) - lacking a definite plan, purpose, or pattern (emphasis added).

The poster stated that the "first four bases were completely random". I assumed (not "presumed") he meant what he wrote :-).

Semantically, "high complexity" does not mean base composition diversity, which is the relevant issue for cluster calling. A library that consists solely of AAAAA, CCCCC, GGGGG, or TTTTT starts (in roughly equal amounts) would suffice, yet (almost) no one would argue that this constitutes high complexity.

Apologies if this message comes across as cranky, Phillip. I was just trying my best to help the poster, and don't see how your comments contribute to the solution.

-Harold

[pmiguel]

If the first four bases are random, then subsequent low complexity should not adversely affect cluster calling or data quality. Excessive cluster density is a possible culprit: what are your raw and PF values?

Semantically "random" does not mean "high complexity". See.

Point being we presume "random" here means all four bases were a random mix of ACGT and therefore an even mixture of all 256 possible sequences. But we would need to know what the method of generating these was to know.

--
Phillip

[greigite]

For what it's worth we sequenced two lanes on a HiSeq (v2 flow cell) containing 11 7 bp inline barcodes with a spike-in of 5% phiX. Despite the low diversity visible in fastqc plots for the first 7 bp our cluster density and % of clusters passing filter was comparable to other lanes on the same run that did not have any low-complexity issues.

[csquared]

We do similar things to what you are describing all the time. A 20-30% PhiX spike (or any other library) should do the trick. PhiX is easy since it can be easily removed without an index and you can monitor the percent alignment as the run is going.

Our most common condition is a HiC or 5C library where we need to get through some T3 and T7 sequences that are common to all of the samples. We have used both ChIPseq libraries as well as PhiX spikes with very good results. The use of the ChIPseq libraries just allows those reads to be used for something useful where PhiX is just data thrown away.

Add in a spike and lower cluster density on the HiSeq to the 500k to 600k range and you should be fine. If you want to avoid the spike altogether, lowering clusters to about 200k also works but with more variable results.

[HESmith]

If the first four bases are random, then subsequent low complexity should not adversely affect cluster calling or data quality. Excessive cluster density is a possible culprit: what are your raw and PF values?

[HMorrison]

There's an alternative approach, assuming that you have not yet constructed the libraries. Design them so the junction is at the opposite end of the insert, and perform paired-end sequencing. Cluster calling is based only on the first five cycles of read one, so you'll avoid the low-complexity issue.

I have a sample of 96-plex low diversity amplicon libraries running now and clusters were found just fine--but the low diversity is causing a tremendous discrepancy between the blue and the green box-and-whiskers plot--raw clusters and clusters passing filter. I hope those data are recoverable at the end. Nothing in my primer design, barcoding, indexing scheme can change the fact that it's "low complexity". First four bases were completely random and followed by eight different in-line bar codes.

This is PE sequencing.

Yet I know labs are making this work.

[pmiguel]

I have seen bad batches of phiX that had fairly high (a few percent, I think) adapter dimers levels in them. Maybe you should make your own genomic DNA library to make sure your "diluent" is of high quality.

Also you could obtain your "diluent" by sub-contracting a sequencing job. Send an email out to a prospective department (maybe one with a high level of plant or fungal sciences being done) and offer a one time only discount genome sequence. Our diluent was a sorghum genomic DNA library.

--
Phillip

[fkrueger]

Just as another thought, if you could afford to spike in 90-95% gDNA, couldn't you also find an external sequencing facility who still run GAIIx's and use the methods which work well on these?

[BIG_SNP]

We have had great success using the NuGen library prep. Their adapters have inline barcodes which adds to the diversity for the first cycles and allows sequences to pass filter. After passing filter the HiSeq can sequence the no or low diversity samples without any problems.

[Simcom]

We got it to work on our HiScanSQ -- which uses the same chemistry as the HiSeq, but only scans the top of the flowcell. Not an identical situation, but we had some SMART cDNAs that we sheared and ligated TruSeq adapters on. So about 1/2 of them had the same 50 nt of SMART primer at the beginning. We mixed them 1:1 with a genomic DNA library. Cluster registration went fine.

--
Phillip

OK, that is good to hear. I'm not sure why my core was having trouble spiking 1:1 gDNA.

[Simcom]

Yes, adapter reads are present.

Just remember that you'll also need to include the standard sequencing primer for the gDNA library (or construct the gDNA library with custom adapters to match your custom primer).

Yep, I planned on including both primers. Thank you so much for your help, I really appreciate it.

[HESmith]

Yes, adapter reads are present.

Just remember that you'll also need to include the standard sequencing primer for the gDNA library (or construct the gDNA library with custom adapters to match your custom primer).

[pmiguel]

We got it to work on our HiScanSQ -- which uses the same chemistry as the HiSeq, but only scans the top of the flowcell. Not an identical situation, but we had some SMART cDNAs that we sheared and ligated TruSeq adapters on. So about 1/2 of them had the same 50 nt of SMART primer at the beginning. We mixed them 1:1 with a genomic DNA library. Cluster registration went fine.

--
Phillip

[Simcom]

If, as you say, you can tolerate discarding 90-95% of the reads, then spiking in a gDNA library at that level will definitely solve your problem. After all, adapter dimers are often present at 5-10% in many libraries (the same % as your desired samples), and sequencing them is not a problem!

Thanks, this gives me confidence. Just to be sure though: do the adapter-adapter ligation reads come back in the data, or does the machine throw them out and not include them in sequencing results? If you actually get the adapter-adapter reads from the machine, I should be golden.

[HESmith]

If, as you say, you can tolerate discarding 90-95% of the reads, then spiking in a gDNA library at that level will definitely solve your problem. After all, adapter dimers are often present at 5-10% in many libraries (the same % as your desired samples), and sequencing them is not a problem!