Maximum read cycles in NextSeq kits?

M4TTN · 12-13-2017, 03:26 AM

Hello,
We are using the 75 cycle 400M kit for paired-end sequencing.
Our understanding is that there are 75 read cycles, plus another 16 cycles (2x8 cycles) available for barcode detection. Total of 91 cycles.
I know that it is possible to split the read cycles into a *minimum* of 2x37. However, our samples are only single-indexed and only use 6 bases for the index.
Thus, in theory, there are another 10-11 cycles of chemistry in these kits, suggesting that 2x42 bp should be possible - plus 6 cycles for indexing.
Does anyone have experience with setting up such a custom run?
Would we simply specify 2x42 bp in the sample sheet?
Many thanks!

GenoMax · 12-13-2017, 04:25 AM

Here is Illumina's official take on the amount of reagents available in all kits.

M4TTN · 12-13-2017, 07:15 AM

Thanks for the link: it looks like 2x42 + 6 = 90 is within range (91 cycles).
I'll report back if there is a problem.

M4TTN · 12-14-2017, 02:16 AM

2x42bp + 6bp index worked perfectly. Approx 190 K/mm clusters, and >95% PF, yielded a total of 480M paired reads. Q-scores look excellent.

I am tempted to try incrementing the read length in successive runs to see at what point the cartridge runs out of reagents...there is bound to be an excess.

GenoMax · 12-14-2017, 05:21 AM

Don't push your luck. If air gets sucked into sippers etc you could ruin something trying to get that extra cycle of sequence.

M4TTN · 12-14-2017, 05:45 AM

Fair comment. Certainly not wanting to upset the machine. I did see a user on here extend the sequencing run on a MiSeq reagent kit and got more than an extra 50 cycles.

We are currently struggling trying to obtain the FASTQs from BaseSpace...they don't seem to be available for some reason on BaseSpace or BaseMount, and requeueing the job has resulted a FASTQ failure message.

We were not the original owners of the run, but ownership was transferred fine (it seemed). Any ideas?

GenoMax · 12-14-2017, 08:23 AM

Contact Illumina tech support for issues related to BaseSpace permissions (techsupport at illumina.com).

JPflueger · 05-31-2018, 07:03 AM

Hey there, just wondering if you ever played around with this to work out how many cycles you could squeeze out of a 75 cycle Nextseq kit?

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12-13-2017, 03:26 AM	#1
M4TTN Member Location: UK Join Date: Jan 2014 Posts: 75	Maximum read cycles in NextSeq kits? Hello, We are using the 75 cycle 400M kit for paired-end sequencing. Our understanding is that there are 75 read cycles, plus another 16 cycles (2x8 cycles) available for barcode detection. Total of 91 cycles. I know that it is possible to split the read cycles into a minimum of 2x37. However, our samples are only single-indexed and only use 6 bases for the index. Thus, in theory, there are another 10-11 cycles of chemistry in these kits, suggesting that 2x42 bp should be possible - plus 6 cycles for indexing. Does anyone have experience with setting up such a custom run? Would we simply specify 2x42 bp in the sample sheet? Many thanks!

12-13-2017, 04:25 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	Here is Illumina's official take on the amount of reagents available in all kits.

12-13-2017, 07:15 AM	#3
M4TTN Member Location: UK Join Date: Jan 2014 Posts: 75	Thanks for the link: it looks like 2x42 + 6 = 90 is within range (91 cycles). I'll report back if there is a problem.

12-14-2017, 02:16 AM	#4
M4TTN Member Location: UK Join Date: Jan 2014 Posts: 75	2x42bp + 6bp index worked perfectly. Approx 190 K/mm clusters, and >95% PF, yielded a total of 480M paired reads. Q-scores look excellent. I am tempted to try incrementing the read length in successive runs to see at what point the cartridge runs out of reagents...there is bound to be an excess.

12-14-2017, 05:21 AM	#5
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	Don't push your luck. If air gets sucked into sippers etc you could ruin something trying to get that extra cycle of sequence.

12-14-2017, 05:45 AM	#6
M4TTN Member Location: UK Join Date: Jan 2014 Posts: 75	Fair comment. Certainly not wanting to upset the machine. I did see a user on here extend the sequencing run on a MiSeq reagent kit and got more than an extra 50 cycles. We are currently struggling trying to obtain the FASTQs from BaseSpace...they don't seem to be available for some reason on BaseSpace or BaseMount, and requeueing the job has resulted a FASTQ failure message. We were not the original owners of the run, but ownership was transferred fine (it seemed). Any ideas?

12-14-2017, 08:23 AM	#7
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,080	Contact Illumina tech support for issues related to BaseSpace permissions (techsupport at illumina.com).

05-31-2018, 07:03 AM	#8
JPflueger Junior Member Location: Australia Join Date: May 2018 Posts: 1	Hey there, just wondering if you ever played around with this to work out how many cycles you could squeeze out of a 75 cycle Nextseq kit?