amplicons

[klymiuk]

Hi users,
doese anyone share our problem with short reads (>80%) during amplicon (with shotgun adapter) sequ and did you find a reason for that? We use highly purified amplicons, so no dimers or sth.
Thanks a lot. Best!

[MissDNA]

Are you using XLR70 or XL+ chemistry?

[pmiguel]

Hi users,
doese anyone share our problem with short reads (>80%) during amplicon (with shotgun adapter) sequ and did you find a reason for that? We use highly purified amplicons, so no dimers or sth.
Thanks a lot. Best!

What evidence do you have that your amplicons are contain no dimers? Very few methods would remove dimer strands that anneal back to the adapters of larger library strands.

What are the length of your amplicons?

--
Phillip

[RCJK]

What signal processing pipeline did you use? Shotgun or amplicon?

[klymiuk]

Hi everybody,
thanks a lot for your questions and comments:
- we are using XLR70 Titanium chemistry
- our amplicons range from 400 - 500 bp, we purify them with gel cut or by HPLC
- we are using the amplicon processing pipeline
thanks a lot for any hint!!
best.

[RCJK]

Do you have a bioanalyzer trace of the amplicons, to help show that there were no small fragments present? They recommend running on a DNA 1000 chip, but lower levels of primers/dimers will show up better on a HS DNA chip.

[pmiguel]

Yes, I see a lot of cases of this type on this forum where it is claimed "there are no small fragments present" and then when they post the bioanalyzer images there are are small fragments present! They are small peaks -- but there is the issue of molarity vs mass here. Even weakly fluorescing low molecular weight peaks may have substantial molar concentration.

--
Phillip

[proteasome]

I don't buy the tired excuse of small fragment problems leading to bad amplicon sequencing. No matter how many times we SPRI purify and no matter how clean our traces look, we have had many months of highly inconsistent and discordant amplicon results, ALL with amplicons and samples that we have previously been able to sequence perfectly.

Why the change? At the risk of being arrogant, I know it is not on us: our staff have not changed. We have had engineers out to look at our machine, we have wasted > $20k in failed reagents. I believe there are production problems with some of the reagents, probably the emulsion oil and maybe the emPCR reagents, that are causing failures.

The lack of QC on the kits we are sent is pushing us away from 454. Unfortunately, switching to Illumina may not be much better if Roche takes them over and they start having the same R&D and production problems.

[klymiuk]

Thats an example of our BA....I have a little bit the assumption of a Roche problem too...but would be very happy for any other hint to solve our problem. What kind of cleanup do you use? What kind of adaptors?....does it work?
best.

[Anthony.287]

And to address the small fragments real quick – we were told that we had them over and over again, until an FAS sat down and looked at the runs with us. What tech support was seeing were reads that were filtered out and included in the short quality category. If I remember which filters are included in this, it’s the Signal Intensity Filter, Valley Filter, and Q-score Trimming Filter. So it seems that a lot more than just real, short, products are included in the short quality filter, which was misleading to us for a long time….

[Anthony.287]

A big, big issue for me is that there has been construction going on directly above and/or below the room with the 454 and BioAnalyzer for the entire time we've had it. I can't really rely on the BioAnalyzer results at all, which is why I run every sample in duplicate, at least.