Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • 16S library preparation - multiple bands

    Hi,

    I am carrying out trial PCRs for 16S amplicon sequencing on Illumina MiSeq. We are targeting the V4 region of 16S using standard 515F/806R primers. Our aim is to profile the microbiome of fish guts under different experimental conditions.

    Our starting PCR protocol is one used by an established high-throughput lab but this generates two distinct bands in certain of our samples (see attached photo). The smaller fragment is ca. 400 bp, which we would expect for the V4 region plus attached primers. The larger and often brighter band is around 600 bp, much bigger than our targeted fragment.

    This larger fragment is not coming from external contamination as both our negative controls and our positive controls (Zymo mock community) do not show this band. The negative controls showing no bands and the positive only the targeted 400 bp band.

    Has anyone seen similar sized bands in their 16S V4 gels? Could it be fish mitochondrial 16S amplification? Would it be 200 bp larger than standard microbial 16S V4?

    Does anyone have advice on how to identify this band and/or how to best optimise my library prep. protocol to remove it, as I don't want to waste sequencing coverage on this artefact. I have tried increasing the annealing temperature and reducing the number of cycles with some positive effect.

    Thanks in advance,

    Alan
    Attached Files

Latest Articles

Collapse

  • seqadmin
    Current Approaches to Protein Sequencing
    by seqadmin


    Proteins are often described as the workhorses of the cell, and identifying their sequences is key to understanding their role in biological processes and disease. Currently, the most common technique used to determine protein sequences is mass spectrometry. While still a valuable tool, mass spectrometry faces several limitations and requires a highly experienced scientist familiar with the equipment to operate it. Additionally, other proteomic methods, like affinity assays, are constrained...
    04-04-2024, 04:25 PM
  • seqadmin
    Strategies for Sequencing Challenging Samples
    by seqadmin


    Despite advancements in sequencing platforms and related sample preparation technologies, certain sample types continue to present significant challenges that can compromise sequencing results. Pedro Echave, Senior Manager of the Global Business Segment at Revvity, explained that the success of a sequencing experiment ultimately depends on the amount and integrity of the nucleic acid template (RNA or DNA) obtained from a sample. “The better the quality of the nucleic acid isolated...
    03-22-2024, 06:39 AM

ad_right_rmr

Collapse

News

Collapse

Topics Statistics Last Post
Started by seqadmin, 04-11-2024, 12:08 PM
0 responses
16 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 10:19 PM
0 responses
21 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-10-2024, 09:21 AM
0 responses
16 views
0 likes
Last Post seqadmin  
Started by seqadmin, 04-04-2024, 09:00 AM
0 responses
46 views
0 likes
Last Post seqadmin  
Working...
X