Using Illumina MiSeq to QC Libraries (NOT LibraryQC module)

sneakers6 · 02-28-2021, 09:54 PM

Hello! First-time poster, but long-time lurker... Thank you for the countless times this forum has aided me in a pinch.

I now turn to a question that has been bugging me lately and I can't figure it out (it's a protocol/strategy question mostly): I run about 200 libraries at a time, and in lieu of doing 19 Bioanalyzer runs to QC each individual library before pooling and sending out to sequence (mostly on a NovaSeq), we had an idea to use our in-house MiSeq to run a quick run to see if the libraries actually succeeded in library prep, and how they are. This saves us time most importantly, which is precious

I would use the LibraryQC module Illumina provides, but it requires a reference genome, and our library pools (of 200 libraries) are typically mixed bacteria communities, typically needing assemblies, so this wouldn't work.

My question is: how would I approach this? I need to be able to use MiSeq FASTQ stats (i.e. indexing QC) to replace the metrics a Bioanalyzer gives you so I can pool these libraries, while doing the best I can to keep the spread of library sequencing as tight as possible and provide pooling info so I can re-pool and adjust if certain libraries need more/less of sequencing depth on the NovaSeq once we send it out.

Thank you in advance!

GenoMax · 03-01-2021, 04:52 AM

You should be able to run MiSeq nano FC (~1 M clusters) to check the balance of libraries and most of all to verify that libraries have worked. We routinely do that for pools destined for NovaSeq.

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02-28-2021, 09:54 PM	#1
sneakers6 Junior Member Location: San Jose Join Date: Feb 2021 Posts: 1	Using Illumina MiSeq to QC Libraries (NOT LibraryQC module) Hello! First-time poster, but long-time lurker... Thank you for the countless times this forum has aided me in a pinch. I now turn to a question that has been bugging me lately and I can't figure it out (it's a protocol/strategy question mostly): I run about 200 libraries at a time, and in lieu of doing 19 Bioanalyzer runs to QC each individual library before pooling and sending out to sequence (mostly on a NovaSeq), we had an idea to use our in-house MiSeq to run a quick run to see if the libraries actually succeeded in library prep, and how they are. This saves us time most importantly, which is precious I would use the LibraryQC module Illumina provides, but it requires a reference genome, and our library pools (of 200 libraries) are typically mixed bacteria communities, typically needing assemblies, so this wouldn't work. My question is: how would I approach this? I need to be able to use MiSeq FASTQ stats (i.e. indexing QC) to replace the metrics a Bioanalyzer gives you so I can pool these libraries, while doing the best I can to keep the spread of library sequencing as tight as possible and provide pooling info so I can re-pool and adjust if certain libraries need more/less of sequencing depth on the NovaSeq once we send it out. Thank you in advance!

03-01-2021, 04:52 AM	#2
GenoMax Senior Member Location: East Coast USA Join Date: Feb 2008 Posts: 7,092	You should be able to run MiSeq nano FC (~1 M clusters) to check the balance of libraries and most of all to verify that libraries have worked. We routinely do that for pools destined for NovaSeq.