[pmiguel]

The arrow is a cartoon of the TCSA left adapter. The orange box denotes the 8 base "i5" index. The green box, as labelled, is some locus specific sequence. The 1st PCR primer fuses the locus-specific sequence with 33 bases of the proximate end of the TCSA adapter. The 2nd PCR primer overlaps the first by 20 bases.

You would also need the right adapter oligos. Basically the same design but with slightly different lengths.

For 96 indexes, you would want 8 i5 indexs and 12 i7 indexes. For 384 you would want 16 and 24, respectively.

I actually screwed up on the right-side oligos and included the reverse complements of the TCSA i7 indexes. But as long as one puts the right sequences in the sample sheet, everything works out okay.

--
Phillip

By the way, I have seen a couple of crazy results when the number of PCR cycles chosen is, well, crazy high. If you had time to optimize, I would suggest trying to use the minimum number of cycles for each PCR that gets you into the 2-10 nM range after clean-up and pooling.

Think about what you are doing! Remember each PCR cycle potentially doubles the amount of the initial template. So, say you are amplifying a 1 kb segment of a 1 billion bp genome. 1 ug of DNA from this organism will contain 1 pg of the segment of interest. How many PCR cycles, theoretically, do you need to obtain enough product?

10 cycles? 1 thousand-gold amplification (pg become ng)
20 cycles? 1 million-fold amplification (pg become ug).
30 cycles? 1 billion-fold amplification (pg become mg -- impossible because your PCR will run out of primers, nucleotides, etc.)

Also, the purpose of the (2nd) step-out PCR is to just add the rest of the adapters -- 3 or 4 cycles should be plenty!

Of course PCR can't achieve a doubling of template concentrations in each cycle -- but this gives you an idea of how heavy the hammer you are smashing your project with!
--
Phillip

[Vinz]

We actually have not tried to merge the bad runs (without hardcoding). But it might be the case, that the Q values are underestimated. The hardcoded runs work well for merging. We have overlaps between 30 and 100bp.
I am pretty sure, that the phiX calculated error rate is a true error rate. I have seen weird spikes in the error rate that did not match the Q-values. Therefore it can not just be cheated Q value display.

[GenoMax]

Question: are your amplicons short enough to overlap the reads? For the run we describe above, the amplicons have 450 bp inserts. So for a paired read merge (Rick uses PANDA, but seems like most people use FLASH), one would expect to need high quality sequence over the entire length of both reads to effect a good merge. However, mysteriously, we had very high rates of successful merges even though the quality drops very low past 180 bases for read 1.

This has been our observation as well. Read overlaps work adequately.

This could be simple a case of the instrument mistakenly assigning low quality values while correctly assigning the base calls.

That is my hunch right from beginning. It seems as if software just gives up on assignment of quality values after it sees consecutive low-nucleotide complexity data for a certain number of cycles.

Strategy described by "BBthekid007" works well. We have successfully used it.

[GenoMax]

The real solution needs to come from Illumina, but they aren't going to bother doing it unless they get enough complaints.

--
Phillip

I can assure you that this is not true. Unfortunately that is all I can say for the moment.

[LVAndrews]

hardcoding question

Thanks for this excellent thread that seems to distill a lot of questions (and answers) to one place. My only question is, do you bother changing out your config.xml file for a genome run? Seems like this wouldn't be necessary at all.

AK

[Vinz]

I definitely would. The caveat with using hardcoded phasing/matrix is, that the instrument does not detect when things change. Eg. a different lot might perform different. Since those values are considered when Q values are calculated, your Q values might be off. So I think, if you have genomic samples in between, you should run them without hardcoding and monitor matrix and phasing values. In case they change, update your hardcoding settings.
Anyone out there has experience how stable those values are?
Do they change more when going from instrument to instrument or when changing lots?

[microgirl123]

The arrow is a cartoon of the TCSA left adapter. The orange box denotes the 8 base "i5" index. The green box, as labelled, is some locus specific sequence. The 1st PCR primer fuses the locus-specific sequence with 33 bases of the proximate end of the TCSA adapter. The 2nd PCR primer overlaps the first by 20 bases.

You would also need the right adapter oligos. Basically the same design but with slightly different lengths.

For 96 indexes, you would want 8 i5 indexs and 12 i7 indexes. For 384 you would want 16 and 24, respectively.

I actually screwed up on the right-side oligos and included the reverse complements of the TCSA i7 indexes. But as long as one puts the right sequences in the sample sheet, everything works out okay.

--
Phillip

Are you actually doing this? I have a great PowerPoint presentation from someone at Illumina that explains how to do a two-step PCR to attach Nextera indices. It's a bit short on details though so I'd love some input with the technical details, like pooling, PCR cleanup, and Qubit vs. KAPA quantification. I've inquired of the Illumina person who sent me the PowerPoint, but it's been over a week and I haven't heard anything. Thanks! Janet

[GenoMax]

Anyone out there has experience how stable those values are?

Short of running another phiX run it would be difficult to know for sure. There has been no indication from illumina that this would need to be checked/changed periodically.

Do they change more when going from instrument to instrument or when changing lots?

Values are definitely different going from one instrument to another. I just checked with the two MiSeq's I have access to.

Have you noticed any changes in Q-values between samples run with hard-coded values in place (and otherwise) for "normal" samples?

We have left the hard-coded matrix file in place and have not seen any obvious problems with "normal" genomic samples. When most of data is > Q35 minor differences in Q values may only be of technical interest.

[pmiguel]

Are you actually doing this? I have a great PowerPoint presentation from someone at Illumina that explains how to do a two-step PCR to attach Nextera indices. It's a bit short on details though so I'd love some input with the technical details, like pooling, PCR cleanup, and Qubit vs. KAPA quantification. I've inquired of the Illumina person who sent me the PowerPoint, but it's been over a week and I haven't heard anything. Thanks! Janet

Did not see this when you first posted it. We have customers who have used method. The one that seemed the best to me, they limited the number of cycles they used -- 15 for the initial amplification, then 5 for the step out PCR step. They used fluorimetry to estimate concentrations of the products. I think they just used a normal PCR clean-up kit.

As with the 454 method, you want to do an ampure clean up on the final pool if you see even a hint of an adapter dimer/primer dimer peak.

--
Phillip

[pmiguel]

I can assure you that this is not true. Unfortunately that is all I can say for the moment.

How very mysterious. I hope this is true.

--
Phillip

[LVAndrews]

Did not see this when you first posted it. We have customers who have used method. The one that seemed the best to me, they limited the number of cycles they used -- 15 for the initial amplification, then 5 for the step out PCR step. They used fluorimetry to estimate concentrations of the products. I think they just used a normal PCR clean-up kit.

As with the 454 method, you want to do an ampure clean up on the final pool if you see even a hint of an adapter dimer/primer dimer peak.

--
Phillip

This was described by Berry et al (2011). If you want to check it out, here's the AEM link: http://aem.asm.org/content/early/201...2/AEM.05220-11

I'm using this for amplicon work on the Miseq since my limited mock community data suggests I get very little skew of abundances compared to say, the EMP protocol (35 cycles, direct tailing). I'm using a thermal profile very similar to the ABI cycle sequencing protocol and getting visible bands after 10 cycles using Phusion HS II polymerase (although the KAPA rep has been pushing me toward their HiFi polymerase). 5 more cycles adds the tails. Use cheapo bead cleanup at any step you choose (http://www.molecularecologist.com/20...re-substitute/) to eliminate those pesky unincorporated primers.

[kmcarr]

Quote:
Originally Posted by GenoMax
I can assure you that this is not true. Unfortunately that is all I can say for the moment.

How very mysterious. I hope this is true.

--
Phillip

Perhaps this thread is a clue.

[GenoMax]

Illumina released a new version of MiSeq software updater yesterday that includes a new version of RTA that helps with low nucleotide diversity samples. Their recommendation is to keep the cluster density in "standard" range (do not over cluster) and add a 5% phiX spike-in.

You can find the installer here: http://support.illumina.com/sequenci...downloads.ilmn

[pmiguel]

Illumina released a new version of MiSeq software updater yesterday that includes a new version of RTA that helps with low nucleotide diversity samples. Their recommendation is to keep the cluster density in "standard" range (do not over cluster) and add a 5% phiX spike-in.

You can find the installer here: http://support.illumina.com/sequenci...downloads.ilmn

Anyone tried it yet? How does it work compared to hard coding?

--
Phillip

[microgirl123]

Where did you find the info about not lowering cluster density and only using a 5% phiX spike? I'd be really reluctant to test this on someone's sample! I have a RADseq library coming up that has some in-line indices that were poorly designed/chosen together with the problem that all the inserts start and end with the same restriction digest sites.