MiSeq cluster generation problems

[skosuri]

After several failed runs in a row (even loading at 50pM), I figured out my problem (with the help of very patient illumina tech support representatives ); so I thought I would update.

I had been using the old P5 and P7 sequences (single end versions, Nature 2008 paper; bentley et al; supp info) for the adaptors I PCR'd on. Apparently these changed for the paired end versions (same sequence with a few extra bases on the 3' end; see the nature 2008 paper).

So while my libraries worked for past GAIIx runs (because both primers were on the flow cells), at some point this changed and the MiSeq flow cells no longer contain these shorter P5/P7 sequences. Thus any clusters I was getting were due to very low levels of mispriming off the forward primer (reverse primer was fine since I am using standard multiplexing primers). Though those misprimed clusters would sequence fine.

Since all the quantification kits have the old P5/P7 sequences, all my RT-PCR titrations looked fine; it was just that the extra bases inhibited most cluster formation of my library.

I reordered the primers now, so I'll re-run everything again, but this explained all my previous problems.

Thanks everyone,
Sri

[ITseq]

Is there heterogeneity for the first 6 bases of your amplicon after your custom sequence primer?

[ECO]

Hmmm...reload that sucker at a higher concentration and see!

I would really love ILMN to facilitate a cheaper/faster way of checking cluster density...

[skosuri]

Does more than 1mM end up hitting the flow cell?

Well it's about 1mM. 10uL in 1mL of 0.1M; this gets diluted slightly, but not much. Though those are the recommendations based on the MiSeq sample prep guide. Plus, the cluster density of the phiX is not affected.

[ECO]

Interesting, but I use the recommended NaOH (0.1M) concentrations.

Does more than 1mM end up hitting the flow cell?

[skosuri]

I would guess it's your molecule lengths, if there is nothing else wrong with the library (see other discussions in the 454 forum about the presence of primer-dimer throwing off qPCR calcs), and your adapter design is sound.

The reads that I do get look good. I don't see many primer dimer reads; everything maps to my library or phiX. The index reads look fine as well. The sequencing results also make me think that the adaptor design works.

Did you denature in a way that would result in higher NaOH concs than recommended? This kills cluster density as well.

Interesting, but I use the recommended NaOH (0.1M) concentrations.

As for the lengths, I sent a similarly sized library for HiSeq sequencing, and the data looked great (300 million good reads in a lane).

[ECO]

I would guess it's your molecule lengths, if there is nothing else wrong with the library (see other discussions in the 454 forum about the presence of primer-dimer throwing off qPCR calcs), and your adapter design is sound. In my very limited experience, clustering efficiency drops off dramatically with molecules <250 and >800. Clustering was much less efficient for 100-120bp amplicons (which end up at ~220 after ligation) than it was for 150-170bp amplicons (~290 after ligation). I'd check for a correlation between #reads and length of amplicon...

To answer your second point, I have never had to go above 11pM with a library pool to get very high cluster densities (>1100-1300). This is with libraries of amplicons (amplicons being 150-170bp before adapters), shotgun libraries, and enriched libraries (both with insert sizes >200bp before adapters).

Did you denature in a way that would result in higher NaOH concs than recommended? This kills cluster density as well.

[skosuri]

Hi all,

I'm running a library of synthetic amplicons (~175-250bp), and reading it out with custom primers on the MiSeq. I'm having a lot of problems getting enough cluster density. My last MiSeq run, I combined the custom sequencing primer with the Illumina Read 1 mix, and ran the mixed 20% 8pM phiX with with 80% 20pM library. I got ~1.5 million phiX reads and ~500K of the custom reads, with at total cluster density on ~225K/mm2. The custom reads looked fine and were what I expected of my library. I reran the Kapa QPCR quantitation again on my dilutions against phiX and it looked pretty accurate.

I was wondering what the concentration range of getting good cluster density is. Would it make sense to run 10X (200pM) of the library to get 10X the number of clusters? This seems ludicrously high. Does it something to do with the length of my library? Has anyone seen such a drastic difference between Kapa Library quantification and cluster density? I'm a little lost and want to figure stuff out without burning too many flowcells.

Thanks,
Sri