Question about wet lab method: "small mean insert size" warning from metagenome assembly

[Rachel Mugge]

Hello,

I am working through an assembly-based method for a metagenome sample (sequence at IMR) following select steps from this tutorial: https://github.com/nikhilg123/Module_5_metagenome_assembly-MAGs/blob/main/Module%205_metagenome_assembly%2BMAGs.md

After the assembly step using Spades (with '--meta' flag), everything went well and produced output, but reported this warning:

"Estimated mean insert size 148.573 is very small compared to read length 151"

I am still able to continue with the output (contigs/scaffolds), but is this warning something to be concerned about? I think the insert size is related to the library preparation step, and the consensus from an internet search seems to be that this is sometimes an issue resulting from wet lab methods rather than the assembler itself. I just want to double check that nothing is inherently wrong with the data and that I can use the assembly output for the next step in the pipeline.

Thank you!

[Andre Comeau]

Yes, it should be OK. The Nextera Flex (now "DNA Prep") kit from Illumina tends to produce fragment sizes more around 250 bp, so the 2x150bp NextSeq we do covers the fragments with a good overlap. However, some DNAs, due to their nature and/or shearing extent/degradation, can sometime produce a peak fragment sizes that are a bit smaller and then the assemblers are also removing the adapter content from the libraries and so sizes can get a bit smaller.

	ANDRÉ M. COMEAU, PhD Manager • Integrated Microbiome Resource (IMR) T: 902.494.2684 | E: andre....@dal.ca	​Address for deliveries: Dept. of Pharmacology Tupper Med. Bldg., room 5D Dalhousie University 5850 College St. Halifax NS B3H 4R2
​	Research Associate (Lab Manager) Morgan Langille Lab • Dept. of Pharmacology ResearchGate Profile • GoogleScholar Publications
"Without fantasy, there is no science. Without fact, there is no art." - Nabokov "The good thing about science is that it's true whether or not you believe in it." - Neil deGrasse Tyson

---

From: microbio...@googlegroups.com <microbio...@googlegroups.com> on behalf of Rachel Mugge <rachel...@gmail.com>
Sent: Wednesday, August 3, 2022 3:27 PM
To: Microbiome Helper <microbio...@googlegroups.com>
Subject: [microbiome-helper] Question about wet lab method: "small mean insert size" warning from metagenome assembly

 

CAUTION: The Sender of this email is not from within Dalhousie.

--
You received this message because you are subscribed to the Google Groups "Microbiome Helper" group.
To unsubscribe from this group and stop receiving emails from it, send an email to microbiome-hel...@googlegroups.com.
To view this discussion on the web visit https://groups.google.com/d/msgid/microbiome-helper/fbb39f54-4dbd-4a8b-8438-8722bd32e90cn%40googlegroups.com.

[Rachel Mugge]

Okay, great- thanks for the quick reply!