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Old 10-17-2017, 01:44 PM   #1
provirus
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Default Making a custom run recipe on nextseq 500

Is it possible to design a complete custom run recipe by yourself on a Nextseq500. For instance I am considering a PE sequence run were some specific cycles are chemistry-only (with no pictures taken) due to some sequence patches in my library with no diversity.
It seems Illumina have no software to support this. They ask one to contact their tech support in order to discuss such possibilities.
While this is of cause the best way to proceed if one like to make a custom run, I am just curious as to whether one could actually make such recipe yourself ? What files are involved in instructing the apparatus and what are the format (etc). Any experience in this from anyone greatly appreciated ?
Thanks.

Last edited by provirus; 10-17-2017 at 11:04 PM.
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Old 10-18-2017, 06:42 AM   #2
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Hi Provirus,

Ask your local FSA if he/she can provide you a custom recipe file with the spec you want. Illumina doesn't advertise it but they gave us the files necessary to run dark cycles (cycles without imaging).
Good luck
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Old 10-18-2017, 10:12 AM   #3
provirus
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Thanks. We will indeed approach Illumina with this. I wanted to know if some users had experience with this, and also to learn about the "inner workings" of the programs that control the runs - it is a black box to me.
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Old 10-18-2017, 11:09 AM   #4
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Been a while since I've done this on the MiSeq, and I know they've consistently updated these files to make it harder to modify...

All the protocols have traditionally been in XML files. So if you go into the Illumina folders on the c: drive, and into the control software folder (not sure what it is called in the NextSeq), under the recipes folder, you should have a subfolders corresponding to various recipes corresponding to preprogrammed methods that pop up in the menu. It's an interesting exercise to understand how they are organized. Frankly, they are messy IMHO...it helps to map out how they are connected.

My sense it that playing with the xml file in the exposure folder would be enough.

But...do you really need to do that? As long as you have diversity in the first 8 (?) bases it should be okay...or, spiking in PhiX at higher concentrations would also be an option.

I may be missing something, though...

Best, Austin
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Old 10-18-2017, 11:41 AM   #5
provirus
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Hi Austin,
interestingly. I will have a look in the folders, and see if it makes any sense. You are right that PhiX spike would help. We are testing this at 35-45% at the expense of library.of-interest - it would be nice if a dark cycles-run would give us more sequence-per-dollar. It's annoying it has to be such a pest to customize runs. (By the way, we have low diversity in the very first cycles - 8 plus some).

Thanks.
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Old 10-18-2017, 12:38 PM   #6
austinso
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Ultimately, the caveat is that the first few bases are used as the QC step to identify successful cluster generation. So having dark cycles may not be in your best interest. But again depends on your application.

I've used 1% phiX for lower diversity runs on the NextSeq (15K primer extension sets), which were fine.

What I've also done in the past is design into the adaptor a mix of pads to offset the reads for extremely low diversity libraries. YMMV of course.
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Old 10-18-2017, 01:37 PM   #7
provirus
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Hi Austin. Is it correctly understood that you had the low diversity regions/patches offset from one another by using 2+ different adapter/amplicon ends (effectively 'moving' the patches relatively to each other) ?
Regards.
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Old 10-18-2017, 03:31 PM   #8
austinso
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Essentially, yes. Either a fixed number of random bases (e.g. 5'-end of primer), or adding 0,+1, +2 bases of known composition.

Quote:
Originally Posted by provirus View Post
Hi Austin. Is it correctly understood that you had the low diversity regions/patches offset from one another by using 2+ different adapter/amplicon ends (effectively 'moving' the patches relatively to each other) ?
Regards.
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Old 10-18-2017, 11:14 PM   #9
JakobHedegaard
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Hi Provirus and Austinso,
What a coincidence. We have just been in contact with Illuminas Techsupport to have a custom program made for the NextSeq 500 to do a PE run with 5 dark cycles after the first 12 cycles : read 1: 151 (1-12 + 13-17 (chemistry-only) + 18-151) cycles read 2 (Index): 6 cycles read 3 (reverse): 151 (1-12 + 13-17 (chemistry-only) + 18-151) cycles.
Would be easier if we could modify the programs, but since we are certified it would be a pain.
/Jakob
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Old 10-19-2017, 12:57 AM   #10
JakobHedegaard
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Hi Provirus,
Please send me a privat message.
/Jakob
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