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Old 11-26-2015, 08:14 AM   #1
Ingeneious
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Default Dark cycles

According to the MiSeq system user guide, the kits contain an additional 26 cycles of reagents (i.e. 50 cycle kit contains 76 cycles of reagents, 300 cycle kit contains 326 cycles of reagents, etc.). Do the 7 dark cycles count against this total?

On a semi-related note, why are the read lengths always N + 1? Will the actual sequencing read(s) actually output this length (i.e. 2 x 150 PE read listed as 2 x 151)?
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Old 11-26-2015, 09:34 AM   #2
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The N+1 has something to do with the phasing. Apparently, for each cycle, the phasing process uses some data from the next cycle, so for the last cycle of the run, there is no N+1 th cycle, and the base qualities are usually rubbish.
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Old 11-26-2015, 11:44 AM   #3
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BBDuk has an optional flag, "ftm=5" (forcetrimmodulo=5), that we always enable for preprocessing raw data. It trims a read so that the length is equal to zero modulo 5. For example, 76 bp reads are trimmed to 75, etc. But if you start with 75 bp reads (which presumably already had the extra 1bp trimmed) then it won't do anything. When used for adapter-trimming, BBDuk trims the extra base prior to trimming adapters, which increases the accuracy due to the very low quality of the extra base.

In practice, you sometimes get 151bp reads and sometimes 150bp from a 2x150 run; I'm note sure why, but it probably depends on either the settings or version of Casava.
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Old 11-26-2015, 01:01 PM   #4
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As @mastal said the last cycle does not have phasing information so generally n-1 cycles are reported by CASAVA or bcl2fastq for tag reads. One can force these programs to output all "n" cycles. So if you absolutely must have "n" bases in tag reads some facilities will run n+1 cycles to get reliable n bases for tags. This does not affect number of cycles for main reads (R1/R2). What you get is how the run was setup.

The dark cycles must count against the total since the instrument is still using reagents for those cycles.

Last edited by GenoMax; 11-26-2015 at 01:04 PM.
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Old 11-30-2015, 09:20 AM   #5
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I do not think dark cycles count against total number of reagents. I could do 72 cycle sequencing (2 x 33 cycles) + 6 cycle barcode = 72 cycles. This is with 50 cycle Illumina kit cartridge.
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Old 11-30-2015, 01:08 PM   #6
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Quote:
Originally Posted by rnaeye View Post
I do not think dark cycles count against total number of reagents. I could do 72 cycle sequencing (2 x 33 cycles) + 6 cycle barcode = 72 cycles. This is with 50 cycle Illumina kit cartridge.
Dark cycles are only used prior to index read #2 in dual indexed runs. There would have been no dark cycles in the setup you described.

The "dark" or chemistry only cycles are used to extend the flowcell grafted P5 oligo the 7 bases needed to reach i5 index sequence.
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Old 11-30-2015, 01:52 PM   #7
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Well, I can tell you that we've run the MiSeq several times with settings that squeeze 175 cycles out of the 150-cycle v3 kit and there haven't been any problems except for a drop in the quality of the very last base, as expected. Likewise 100 cycles out of a 75-cycle NextSeq v2 kit, although I've only seen that once and it was done by a remote service center.

EDIT: based on the next reply, I realize I should specify this was with single-indexing (169 read + 6 index). It sounds like our reads would have had to be a lot shorter if we'd used dual-indexing (152 read + 8 index1 + 8 index2 + 7 dark). I don't like dual-indexing.

Last edited by jwfoley; 11-30-2015 at 02:08 PM.
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Old 11-30-2015, 02:01 PM   #8
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Quote:
Originally Posted by kmcarr View Post
Dark cycles are only used prior to index read #2 in dual indexed runs. There would have been no dark cycles in the setup you described.

The "dark" or chemistry only cycles are used to extend the flowcell grafted P5 oligo the 7 bases needed to reach i5 index sequence.
Thanks for correcting kmcarr. It was an incomplete post by me.
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Old 11-30-2015, 02:09 PM   #9
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Quote:
Originally Posted by jwfoley View Post
Well, I can tell you that we've run the MiSeq several times with settings that squeeze 175 cycles out of the 150-cycle v3 kit and there haven't been any problems except for a drop in the quality of the very last base, as expected. Likewise 100 cycles out of a 75-cycle NextSeq v2 kit, although I've only seen that once and it was done by a remote service center.
All Illumina sequencing kits (HiSeq SBS or MiSeq cartridges) include sufficient reagents for an additional 25 cycles beyond what their stated size is (e.g. 150 cycle kits have reagents sufficient for 175 cycles, 200 cycle have 225 worth, etc.). This is done to provide enough reagents so that you can sequence the full length designated by the kit (including one additional cycle on each read) plus the longest indexing supported by Illumina library kits (dual 2x8bp) and the required dark cycles. Thus a "200" cycle kit will be able to do 2x100bp PE with dual indexs:

Code:
Read 1   101 cycles
Index 1    8
Dark       7
Index 2    8
Read 2   101
============
Total    225 cycles
Of course you would be free to use up those 225 cycles worth of reagents in any format you like, provided the instrument software will accept your custom configuration.
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Old 11-30-2015, 02:13 PM   #10
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Quote:
Originally Posted by kmcarr View Post
Of course you would be free to use up those 225 cycles worth of reagents in any format you like, provided the instrument software will accept your custom configuration.
Yes, N + 25 is the pattern we found in the manual, though it looks like they might be removing that information in more recent editions. And regarding the software, it seems to be getting more "user-friendly" all the time, unfortunately. So get your bonus cycles while you still can!
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Old 11-30-2015, 02:22 PM   #11
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Quote:
Originally Posted by jwfoley View Post
Yes, N + 25 is the pattern we found in the manual, though it looks like they might be removing that information in more recent editions. And regarding the software, it seems to be getting more "user-friendly" all the time, unfortunately. So get your bonus cycles while you still can!
It just occurred to me that we recently ran some custom amplicon libraries submitted by a researcher in which they used dual 12 bp indexes. It was a PE250 run so that is 502 cycles (251+251) plus the indexes (24) and the 7 dark cycles which comes out to 533 cycles. This was run with a MiSeq v2 500 cycle cartridge. The software didn't complain at all about this configuration and we got good data all the way out to the end.
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Old 12-01-2015, 04:12 AM   #12
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Slightly off-topic but does anyone have first-hand experience of what happens when the reagents are exhausted and sequencer starts sucking in air (or is there a fail safe built in that stops sequencing)?
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Old 12-01-2015, 06:10 AM   #13
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There's no fail-safe (at least not on the HiSeq), so it pumps air and the cycle fails (blank images, Q30 plummets to 0).
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Old 12-02-2015, 01:57 AM   #14
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Thanks for all the informative replies. Dark cycles - will try pushing the limit, if I ever need to in the future. I have gone over the 25 extra cycles before and did not have noticeable issues. Phasing for the N+1 cycle makes sense.
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