Seqanswers Leaderboard Ad

Collapse

Announcement

Collapse
No announcement yet.
X
 
  • Filter
  • Time
  • Show
Clear All
new posts

  • 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.

    Comment


    • #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.

      Comment

      Latest Articles

      Collapse

      • seqadmin
        Best Practices for Single-Cell Sequencing Analysis
        by seqadmin



        While isolating and preparing single cells for sequencing was historically the bottleneck, recent technological advancements have shifted the challenge to data analysis. This highlights the rapidly evolving nature of single-cell sequencing. The inherent complexity of single-cell analysis has intensified with the surge in data volume and the incorporation of diverse and more complex datasets. This article explores the challenges in analysis, examines common pitfalls, offers...
        06-06-2024, 07:15 AM
      • seqadmin
        Latest Developments in Precision Medicine
        by seqadmin



        Technological advances have led to drastic improvements in the field of precision medicine, enabling more personalized approaches to treatment. This article explores four leading groups that are overcoming many of the challenges of genomic profiling and precision medicine through their innovative platforms and technologies.

        Somatic Genomics
        “We have such a tremendous amount of genetic diversity that exists within each of us, and not just between us as individuals,”...
        05-24-2024, 01:16 PM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, 06-14-2024, 07:24 AM
      0 responses
      12 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 06-13-2024, 08:58 AM
      0 responses
      14 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 06-12-2024, 02:20 PM
      0 responses
      17 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 06-07-2024, 06:58 AM
      0 responses
      186 views
      0 likes
      Last Post seqadmin  
      Working...
      X