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I have heard a rumour that the long standing MiSeq v3 600 bp kit chemistry problems which led to poor quality read 2 data have recently been solved by Illumina. Anyone know if this is true?
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It is true and for high diversity libraries output is according to specification. Amplicon sequence quality also has improved but as before cluster density should be reduced and spiked in with PhiX. -
I just started my first 600-cycle run in over a year so hopefully it's true! I'll try to update in a couple of days when it's finished.Comment
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Can anyone confirm this? I need to do some 2x300 sequencing for Ig RNA-seq experiments. Would be awesome if I could use the MiSeq.
Thanks,Comment
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Yes, the last few 2x300 runs were way better than 1 year ago.
Yes, the last few runs were way better (touch the wood).
If doing amplicons, please use a lot of phiX or shotgun library (up to 20%) (esp. if fist 25 bp's are the same). It would not hurt, and gains in data quality/yield from better dephasing calculation far outweigh the losses from 5%-20% phiX sequence spike in.
If doing cDNA amplicon analysis: make sure you do not use oligoC oligos in your amplicons, because the read quality would go through the floor if there are 10-14C's or G's in a row, and you may also get non-specific amplification of rRNA (rcDNA), reducing usable data yield by 1-2 orders of magnitude.
Also lower the loading density for 500-600bp amplicons, if you need high quality reads for your analysis.
Obviously do not forget, that the longer the amplicon or the read is, the more sensitive it becomes to the DNA sequence content.Comment
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Are there alternatives other than Sanger? Or, what was your backup plan?Originally posted by plyguy69 View Post
@Markiyan - thanks for the advice, I'll pass it on!Comment
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Thanks to everyone for their positive replies, that is very good news. We have ordered up a 600 bp kit and will test it ourselves soon and let you know how we get on.Comment
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550bp+ full amplicon sequencing platforms...
1. Pacbio RSII CCS - should work quite nicely for amplicons up to 2-4kb.Originally posted by Brian Bushnell View Post
2. If you can cope with 2-4% systematic errors - Oxford Nanopore 2D.
3. There used to be Roche's systems - FLX+ and Junior, but the reagents for them are no longer available... :-(Comment
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Not good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!Comment
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Oh dear! That's unfortunate, sorry to hear that.Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
Anyone else with recent experience running the 'new' 600 bp kits? We plan to use one this week or next....Comment
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Am curious about the focus on the Q-scores. Is the investigator looking to call SNPs? Aligning to reference sequence should be no problem irrespective what the Q-score is.Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!Comment
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Adapter dimers?
This looks like issues with adapter dimers to me - what was yours beads ampure cleanup ratios, and any slight bump up on the agilent trace in the region of 100-200bp?Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically.
Basically significant amount of the clusters on the run contained shorter templates, and once the end of these is reached (140-160 bp), it wrecks RTA dephasing calculations.
since templates in the 150-200 bp cluster very efficiently compared to 500-800 bp ones, it takes only 3-5% of the shorter contaminant to wreck Q scores in the run.
To minimize it avoid repetitive freeze thaw cycles of the Illumina adapters (they can lose T tails and self ligate) and make sure no nucleases contamination is present during the ligation.Comment
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Tech support has told me not to cluster below ~750k even for v2 even for amplicon runs. If it's 16S, I had a particular set of samples from some sort of bird guts that I basically can't run by themselves even with 20% phiX because R2 is so bad. There seems to be something happening between cycles 10-30 of R2 that is actually sequence dependent. The way my facility is set up, I mix and match small projects on runs so this is addressable by splitting up the bird samples across many runs.Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!
The suggestion that there may be a primer diamer issue is reasonable, but you also may need to up your density a bit.Microbial ecologist, running a sequencing core. I have lots of strong opinions on how to survey communities, pretty sure some are even correct.Comment
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In addition to above comments it could be a bad batch as well. My current runs Q30 are above %70 specified by Illumina for 2x300 reads:Originally posted by microgirl123 View PostNot good
It was an amplicon run, but the cluster density was good (~600K) with 20% phiX, so I expected the Q30 to be much better than 64% overall. It was only 54% for Read 2. Both reads only got out to about 150 bp before dropping off dramatically. The investigator decided it was definitely not worth trying to use the 600-cycle kit again!Comment
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