Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • low R1/R2 (Illumina) overlapping

    Dear experts, I performed a paired-end amplicon (16S V3/V4 region) sequencing (2x250) on Miseq (Illumina) and the trimmed reads present a low (20%) overlapping between R1 and R2. I use this approach oftenly without problems. Any idea about what happened or how can I treat this data? Thanks in advance.
    Last edited by LFMar; 03-11-2016, 07:40 AM.

  • #2
    I guess quality of reads 3’ end (more likely Read2) was low so they have been trimmed and the length is not enough for overlap. You may try merging first and then filter low quality reads or lower trimming stringency.

    Comment


    • #3
      Originally posted by nucacidhunter View Post
      I guess quality of reads 3’ end (more likely Read2) was low so they have been trimmed and the length is not enough for overlap. You may try merging first and then filter low quality reads or lower trimming stringency.
      Agreed. I think the best approach is usually to merge the raw (or adapter-trimmed) reads, then optionally try quality-trimming and merging the remaining unmerged pairs. BBMerge does this internally and it substantially improves the merge rate as opposed to only merging raw reads or only merging trimmed reads.

      In cases like these it's also useful to explain for the forum what kind of data you have and how you did the trimming, by the way. Quite often people trim to extremely high levels like Q25, which is a bad idea, particularly for low-diversity amplicons which often have very low quality.

      Depending on the dataset, it's also possible to error-correct rather than quality-trimming, or even extend non-overlapping reads so they overlap. This is mainly useful for randomly-sheared data, though, and not applicable to amplicons.

      Comment

      Latest Articles

      Collapse

      • seqadmin
        Essential Discoveries and Tools in Epitranscriptomics
        by seqadmin


        The field of epigenetics has traditionally concentrated more on DNA and how changes like methylation and phosphorylation of histones impact gene expression and regulation. However, our increased understanding of RNA modifications and their importance in cellular processes has led to a rise in epitranscriptomics research. “Epitranscriptomics brings together the concepts of epigenetics and gene expression,” explained Adrien Leger, PhD, Principal Research Scientist on Modified Bases...
        Today, 07:01 AM
      • 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

      ad_right_rmr

      Collapse

      News

      Collapse

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