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  • Potential issues with larger insert sizes (450bp) for 2x150bp Illumina sequencing

    Hello,

    We have prepared some metagenomic sequencing libraries using the NEBNext® Ultra™ II FS DNA PCR-free Library Prep Kit for Illumina kit in which we chose an insert size of 450bp. We will be sequencing with NovaSeq X Plus 2x150bp paired-end. We are worried that our long insert sizes will leave too large of an inner distance between the two reads ultimately resulting in poor sequencing quality. I am aware of literature reporting both benefits (https://bmcgenomics.biomedcentral.co...64-022-08316-y) and limitations (https://www.nature.com/articles/s41598-019-39076-7) or longer insert/fragment sizes.

    Does anyone have any experience/advice on whether these libraries with longer insert sizes (450bp plus adapters/indexes) will pose an issue for 2x150bp sequencing?

    Thanks!

  • #2
    Our group routinely prepare libraries with insert sizes of 550 bp. Your sequencing quality will not be impacted, but , as you've noted, the gap between the reads will be larger which shouldn't be an issue. However, you may have to optimize the loading concentration for these libraries due to the larger size of the fragments.

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    • #3
      Dear robertsr,

      I don't think that 450 bp of insert size will be an issue, as long as you're not interested in the whole insert sequence. For ATAC-seq, we routinely prepare libraries with insert sizes ranging from 100 bp to 2 kb and as long as you're careful with libraries quantification, equimolar mix and loading concentration, everything should be fine.

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