Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Opinion on FastQC output for HiSeq 4000 PE sequencing run

    Hello,

    I recently had four 400bp insert plant DNA libraries sequenced (2x150bp) using one HiSeq 4000 lane.

    I've attached FastQC outputs for R1 and R2 of one of these libraries.

    It seems like their are some issues with low-flow sections on the cell(?). R2 reads are noticeably lower quality than the R1 reads for all libraries.

    ~94% of reads from the flow cell remain after deduplication with bbmap (clumpify).

    ~62% of deduplicated reads remain after quality triming with trimmomatic (LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:100).

    There seems to be some remaining adapter in some of the library reads.

    Overall I've ended up with about 235,011,160 read pairs from the whole lane after deduplicating and trimming.

    Other info: library preparation was PCR-free using physically sheared DNA; Inserts were sized by gel purification; Libraries were dual indexed.

    My questions are:

    1. Is this quality/quantity typical from a commercial provider for this platform?
    2. Is the sequence bias observed at the 5' end of the reads observed in these libraries typical for a PCR-free library generated from physically sheared fragments?

    Any additional comments appreciated.

    Thanks in advance
    Attached Files
    Last edited by quokka; 07-30-2018, 06:45 PM. Reason: remove identifying information from attachments

  • #2
    An answer to this question on biostars

    An answer to this question on biostars:

    Comment


    • #3
      The 5' bias is mostly due to the A-tailing reaction.

      Comment

      Latest Articles

      Collapse

      • 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
      • seqadmin
        Techniques and Challenges in Conservation Genomics
        by seqadmin



        The field of conservation genomics centers on applying genomics technologies in support of conservation efforts and the preservation of biodiversity. This article features interviews with two researchers who showcase their innovative work and highlight the current state and future of conservation genomics.

        Avian Conservation
        Matthew DeSaix, a recent doctoral graduate from Kristen Ruegg’s lab at The University of Colorado, shared that most of his research...
        03-08-2024, 10:41 AM

      ad_right_rmr

      Collapse

      News

      Collapse

      Topics Statistics Last Post
      Started by seqadmin, Yesterday, 06:37 PM
      0 responses
      8 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, Yesterday, 06:07 PM
      0 responses
      8 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-22-2024, 10:03 AM
      0 responses
      49 views
      0 likes
      Last Post seqadmin  
      Started by seqadmin, 03-21-2024, 07:32 AM
      0 responses
      67 views
      0 likes
      Last Post seqadmin  
      Working...
      X