Announcement

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

  • 16S library preparation - multiple bands

    Hi,

    I am carrying out test PCRs for 16S amplicon sequencing on Illumina MiSeq. I am using standard V4 primers: 515F/806R. The aim is to profile the microbiome of fish guts under different experimental treatments.

    Gels loaded with product from these test PCRs show two clear bands (see attached photo). The smaller band at 400 bp is what I would expect for the fragment size of the V4 region plus attached primers. The larger and often brighter band is ca. 600 bp in size. This is not likely due to external contamination as both the negative and positive controls (Zymo mock community) did not show this band. Negative controls showing no band and the positive only the expected 400 bp band.

    Has anyone seen similar sized band in their 16S V4 PCRs? Could this be amplification of fish mitochondrial 16S?

    Does anyone have advice on how to control for this non-specific band during PCR and/or how to identify it? I don't want to waste sequencing coverage on a potential PCR artefact.

    Thanks in advance,

    Alan
    Attached Files

  • #2
    To follow-up on my previous question, we TA cloned and then Sanger sequenced the 600 bp fragment. Blast searches of the sequenced fragment came out with the 18S sequence of a congener as the top hit.

    Comparison of the 16S V4 primer sequences with the 18S sequence, reveals a high degree of overlap. So, I think we can, with a degree of certainty, say that the 600 bp band was caused by non-specific amplification of our host species' 18S. Whilst these 18S sequences would be removed during our bioinformatic analysis pipeline, we would rather not waste sequencing 'effort' on this fragment. So the next step will be to develop a blocking primer for 18S, preventing its amplification during PCR.

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Advanced Tools Transforming the Field of Cytogenomics
      by seqadmin


      At the intersection of cytogenetics and genomics lies the exciting field of cytogenomics. It focuses on studying chromosomes at a molecular scale, involving techniques that analyze either the whole genome or particular DNA sequences to examine variations in structure and behavior at the chromosomal or subchromosomal level. By integrating cytogenetic techniques with genomic analysis, researchers can effectively investigate chromosomal abnormalities related to diseases, particularly...
      09-26-2023, 06:26 AM
    • seqadmin
      How RNA-Seq is Transforming Cancer Studies
      by seqadmin



      Cancer research has been transformed through numerous molecular techniques, with RNA sequencing (RNA-seq) playing a crucial role in understanding the complexity of the disease. Maša Ivin, Ph.D., Scientific Writer at Lexogen, and Yvonne Goepel Ph.D., Product Manager at Lexogen, remarked that “The high-throughput nature of RNA-seq allows for rapid profiling and deep exploration of the transcriptome.” They emphasized its indispensable role in cancer research, aiding in biomarker...
      09-07-2023, 11:15 PM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 09-29-2023, 09:38 AM
    0 responses
    12 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 09-27-2023, 06:57 AM
    0 responses
    13 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 09-26-2023, 07:53 AM
    0 responses
    30 views
    0 likes
    Last Post seqadmin  
    Started by seqadmin, 09-25-2023, 07:42 AM
    0 responses
    18 views
    0 likes
    Last Post seqadmin  
    Working...
    X