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Old 01-06-2016, 07:56 AM   #1
buckiseq
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Smile Hello SEQanswers! (MiSeq/NextSeq)

Hello!

I currently work at a laboratory helping to design, develop, validate, and run Next-Gen panels. We started with a MiSeq and have moved to a NextSeq, both using a Fluidigm based library prep. The transition was... interesting. I have used a couple other library prep systems as well.

I look forward to being able to participate in and hopefully contribute to the community in a meaningful way!

Alex
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Old 02-25-2016, 03:54 PM   #2
imd
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Default interesting

So our group just started using fluidigm-based libs on nextseq too and I was wondering if you experienced any problems - especially with the quality of the second read.
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Old 02-25-2016, 06:54 PM   #3
luc
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Fluidigm AccdessArray libraries or Fluidigm C1?
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Old 02-26-2016, 01:44 PM   #4
imd
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AccessArray
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Old 02-29-2016, 06:26 AM   #5
buckiseq
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Default Library type, Sequencing Primers, etc. ?

imd,

Sorry for the delay in my response. Read quality, particularly read two, was among the several problems when making the transition from the MiSeq to the NextSeq. Read two has been consistently worse than read one, but the overall %Q30 difference between the two reads is not too much more pronounced that it was with the MiSeq. One "advantage" of the NextSeq's two color approach is that you can see clusters "drop out" at the end of reads due to dark cycles being interpreted as Gs. We always see an increase in percent G in the last ~40 cycles of read two, sometimes it exceeds 40 or 45% G! We have a very diverse library that should stay at 25% of each base throughout the reads. Are you seeing this? What exactly about quality of the second read is bad?

Could you tell me more about your situation?
  • What is your read length?
  • Are you using Fluidigm's "FL1/FL2" sequencing primers? (We actually had to change our read one seq primers from what we had been using with the MiSeq, per recommendation by Fluidigm. They said and we observed that this increased the quality of the "cluster template" that is used by the system for both reads.)
  • Have you sequenced the same samples and/or panels on another sequencer (MiSeq)?
  • What kind of library is this? Targeted resequencing? RNAseq?
  • How diverse is your library?
  • What % PhiX are you using?

For comparison to your libraries, we run a couple of targeted resequencing panels with over 300 amplicons each. We use the 48x48 access array system. Our samples are FFPE, blood, and bone marrow. We perform 151x151 reads with 10% PhiX.

Fluidigm's support on this was not stellar, but we were both admittedly new to the NextSeq system. Let me know more about your situation or if you have any further questions ours and maybe we can both benefit from improved NextSeq data!

Thanks!

Last edited by buckiseq; 03-08-2016 at 10:19 AM. Reason: Name removal
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Old 03-09-2016, 04:23 AM   #6
imd
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Sorry, I was waiting for some details from the sequencing group - I'm the bioinformatics guy.

So the reads were 150bp paired-end. The same sequence was also run on a MiSeq with no problems. We also changed the read one primer (to something that was longer by about 17 nucleotides - FL1). This improved the quality of read one but read two is still poor quality.

We are using FL1 and FL2. We had also used Illumina seq primers to sequence PhiX (5%) but have now been suggested by Fluidigm to try not mixing Illumina and Fluidigm primers. We'll see if that makes a difference.

We have been doing targetted reseq of an exome lib and an RNASeq lib.

One other strange observation is that we have seen twice as many reads in lanes 1 and 3 compared to 2 and 4. No idea what that means.

What primers are you using and have you seen any difference by not including PhiX.
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Old 03-09-2016, 08:19 AM   #7
buckiseq
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imd, no worries! I forgot to mention that we started by using the v1 NextSeq kits as v2 was not for sale yet, but we switched to the v2 NextSeq kits and had much better success all around. We also had some odd data patterns between lanes 1+3 vs 2+4 using the v1 kits. Illumina called this he "lane effect" and did not offer much more help on that, especially since we were using Fluidigm libraries. The v2 kits appeared to help that, although

Which version of NextSeq reagents are you using?

We did not see a difference when we used the custom ports without the Illumina primers or a PhiX spike, so we elected to keep them and PhiX as a control for whether or not we have a bad cartridge. The % PhiX we see also allows us to infer a bit about whether our sample library was over/under quantified.

We are using one of the "longer" FL1 primers too, but we also "split" the FL1 primer set up. This was partially due to our troubleshooting, so I do not know if splitting them helps, but if you do not then you have a primer in each read that is NOT being used. FL1 contains both your insert read one and insert read two primers, CS1 (now LCS1 that we use for NextSeq) and CS2, respectively. For the NextSeq, all sequencing primers are at 0.30 ÁM final concentration in the cartridge. What primer concentration are you using? We use the following primers for the following reads:

LCS1 - Insert Read 1
CS2 - Insert Read 2
CS2rc - Index read

And as you can see, we also "split FL2" for the index read as we only use one of the primer pairs that composed FL2, since it contained primers for BOTH indices of a dual indexed library, and we only have single indexed libraries. How many indices are you using?

Additionally, the LCS1 primer contains NO LNAs, while the CS2 and CS2rc primers are the original design from Fluidigm that we used in the past, which contain LNAs.

Thanks!

Last edited by buckiseq; 03-09-2016 at 08:23 AM. Reason: Added words for clarification.
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Old 03-09-2016, 09:03 AM   #8
luc
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Hi Buckiseq,

where could I find information on the LCS1 - Insert Read 1 primer? I can only find CS1 in the fluidigm manual and that is what we ordered.
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Old 03-11-2016, 07:10 AM   #9
buckiseq
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Quote:
Originally Posted by luc View Post
Hi Buckiseq,

where could I find information on the LCS1 - Insert Read 1 primer? I can only find CS1 in the fluidigm manual and that is what we ordered.
luc,

Illumina should be able to provide you with the sequence- I am not sure about distributing it myself. We contacted Illumina about our issues with Fluidigm AA libraries on the NextSeq and they gave us "LCS1" and LCS2" primer sequences. We could not use the LCS2 primer since the extra bases "run into" our barcodes, but the improved initial cluster template generated using LCS1 provided better overall sequencing, even though we still used the same CS2 insert read 2 primer from Exiqon (with LNAs, per Fluidigm SOP). PM if you would like to discuss this further.

Do you mind me asking what kind of libraries you are sequencing and if you are switching from a MiSeq to a NextSeq?

Thanks,
buckiseq
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Old 06-23-2016, 02:48 PM   #10
Nepomuk
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Hi all,
hmmmmmm we have absolutely no problems with putting NextSeq libs on the MiSeq for titration (all made with illumina or NuGEN kits) . . . . why are the Fluidigm libraries that strange. Please help me understanding. If they work on the MiSeq shouldnt they work on the NExtSeq . - . - and why then only not on the reverse read?
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Old 06-23-2016, 03:05 PM   #11
buckiseq
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Quote:
Originally Posted by Nepomuk View Post
Hi all,
hmmmmmm we have absolutely no problems with putting NextSeq libs on the MiSeq for titration (all made with illumina or NuGEN kits) . . . . why are the Fluidigm libraries that strange. Please help me understanding. If they work on the MiSeq shouldnt they work on the NExtSeq . - . - and why then only not on the reverse read?
We also have no problem with putting other libraries on both sequencers. The Fluidigm libraries were tough because they require a different sequencing primer from what is in the cartridges (Illumina's sequencing primer). This is due to Fluidigm's "common sequence" tags that flank the sequencing target primers. When we tried using the same Fluidigm sequencing primer on the NextSeq as we did with the MiSeq, these sequencing primers apparently did not work very well with the NextSeq's chemistry. Remember, while the two instruments are both using Illumina's SBS technology, the MiSeq has a reagent chiller and the NextSeq does not - this alone is an indication that there are some chemical differences. Once we adjusted the melting/annealing temp of the sequencing primers used for our Fluidigm libraries, they performed much better.

Does this make sense? Let me know if you have any questions.

Thanks!
buckiseq
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