We are using the 500 cycle kits for amplicon sequencing with about 20 different amplicons. After applying hardcoded settings it looks ok now. Not as nice as a phiX. But we get usually above 75%Q30. The last 50 bases of the reverse read quality drops (20 to 50% below Q20).
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Originally posted by pmiguel View PostAlso, it was my understanding that the new MCS version 2.1.1.13 had some improvements for handling low diversity data. Anyone seen evidence of this?
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Phillip
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Originally posted by Vinz View PostWe are using the 500 cycle kits for amplicon sequencing with about 20 different amplicons. After applying hardcoded settings it looks ok now. Not as nice as a phiX. But we get usually above 75%Q30. The last 50 bases of the reverse read quality drops (20 to 50% below Q20).
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We use the 4 primer approach with dual indexing (see this post). Meaning, we do only 1 PCR which includes a target specific PCR including universal adapters and outer primers which will add the indexes and flow cell sequences. I guess it depends on how many different amplicons you want to target. If you have a low level of targets and multiplexing, then your approach might be worth while.
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Originally posted by Vinz View PostWe use the 4 primer approach with dual indexing (see this post). Meaning, we do only 1 PCR which includes a target specific PCR including universal adapters and outer primers which will add the indexes and flow cell sequences. I guess it depends on how many different amplicons you want to target. If you have a low level of targets and multiplexing, then your approach might be worth while.
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Originally posted by AKrohn View PostThat's fantastic. Exactly what I've been looking for. We're new to providing miseq service and it seems like everyone wants a different locus (which I totally understand).
PCR1F TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGNNNNNNNNNNNNSEQUENCESPECIFICPRIMER
PCR1R GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGNNNNNNNNNNNNSEQUENCESPECIFICPRIMER
PCR2F AATGATACGGCGACCACCGAGATCTACAC[i5]TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG
PCR2R CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG
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Originally posted by AKrohn View PostI had thought this as well, but my first amplicon run after the update didn't seem too great. I asked our FAS and she said it was mainly meant to prevent the intensity spikes that result from high phasing/prephasing values, not to really address the low-complexity problem.
Colour correction is now also estimated over more cycles too making the matrix a bit more robust.
Our area rep presented some data on this recently with a 900k/mm2 loading density, 90% PF with <5% PhiX. It comes with the warning that this isn't a magic fix. It still depends a little bit on what's exactly in your library but it does seem to improve data quality for single amplicon libraries.
We're yet to give it a go. We're thinking of starting with a 15% PhiX spike-in and titrating down as we go.Attached Files
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I have not tried nextera style sequencing primers. All I can say is that until the indexes the sequence looks good.
Our attempts with 12 Ns were not too encouraging. We had the impression that introducing the 12 Ns resulted in more artefacts (short dimers).
Anyway, as Tony stated, the new software works really well. We spike in around 10% phiX with good success. Hardcoded phasing/matrix does not seem to be required anymore. So I would not recommend the 12 N approach.
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You guys should really try phase-shifting-N primers. Have a few sets of primers with varying numbers of N before your amplicon (N, NN, NNN). This gets the identical regions of each sample out of sequence phase with each other. Works well for us so far.
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