Seqanswers Leaderboard Ad

Collapse
X
 
  • Filter
  • Time
  • Show
Clear All
new posts
  • Ann235
    Junior Member
    • Mar 2013
    • 2

    Hello from a Sanger sequencer

    Hi,
    My name is Ann, and I'm an old Sanger sequencer getting used to the NGS world. I used to do radioactive (S-35 and P-33) sequencing, ~3-5 days hands on for 750 bp, so the switch to capillaries was incredible then and the switch to millions of reads makes my younger self green with envy. I've always been a mix of molecular biologist with super-user: I build my own Illumina libraries but send them off to a sequencing core and I can work in the Galaxy Web based interface and can explain what I want to a programmer but I cannot program myself. (I tried. I took a series bioinformatics courses including C++and Perl but I'm so slow at programming that I could calculate an e-value by hand faster than I could code it.)

    Enough about me. I have several questions.

    1) I'm curious why Phred values don't seem to be used by de novo NGS sequencing assemblers, or have I just not read the documentation correctly? They seem only to be used to evaluate whether a MiSeq or HiSeq run was good and not to mask or trim individual bases that aren't good. It seems weird to me to throw out an entire read if only few bases are bad.

    2) What are the average %>Q30 for MiSeq and HiSeqs? Normal ranges?
    I realize that longer reads have declining Q30s, but on my first 2 MiSeq runs, as quality controls before HiSeq runs, at 2 x 150s with 96 combinations of dual index adapters, the core told me I had Read 1 96%>Q30 and Read 4 (Read 2 sequencing primer 2) 92%>Q30 with ~12 million paired end reads PF. On my second run, with 1 index, I had 2 x 250 at about the same # of reads ~94% and ~90% respectively. They said those values were high, I'm just trying to get a feel for how high. The Sequence Analysis Viewer shows that most of the cycles are well above Q35 with the 2 index sets (8 cycles each index Read 2 and 3) in the middle dropping to Q30.

    Thanks in advance,
    Ann
  • JackieBadger
    Senior Member
    • Mar 2009
    • 385

    #2
    Originally posted by Ann235 View Post
    Hi,


    1) I'm curious why Phred values don't seem to be used by de novo NGS sequencing assemblers, or have I just not read the documentation correctly?
    Lots of assemblers. Most use Phred scores in assembly and SNP ID

    Comment

    Latest Articles

    Collapse

    • seqadmin
      Pathogen Surveillance with Advanced Genomic Tools
      by seqadmin




      The COVID-19 pandemic highlighted the need for proactive pathogen surveillance systems. As ongoing threats like avian influenza and newly emerging infections continue to pose risks, researchers are working to improve how quickly and accurately pathogens can be identified and tracked. In a recent SEQanswers webinar, two experts discussed how next-generation sequencing (NGS) and machine learning are shaping efforts to monitor viral variation and trace the origins of infectious...
      03-24-2025, 11:48 AM
    • seqadmin
      New Genomics Tools and Methods Shared at AGBT 2025
      by seqadmin


      This year’s Advances in Genome Biology and Technology (AGBT) General Meeting commemorated the 25th anniversary of the event at its original venue on Marco Island, Florida. While this year’s event didn’t include high-profile musical performances, the industry announcements and cutting-edge research still drew the attention of leading scientists.

      The Headliner
      The biggest announcement was Roche stepping back into the sequencing platform market. In the years since...
      03-03-2025, 01:39 PM

    ad_right_rmr

    Collapse

    News

    Collapse

    Topics Statistics Last Post
    Started by seqadmin, 03-20-2025, 05:03 AM
    0 responses
    49 views
    0 reactions
    Last Post seqadmin  
    Started by seqadmin, 03-19-2025, 07:27 AM
    0 responses
    57 views
    0 reactions
    Last Post seqadmin  
    Started by seqadmin, 03-18-2025, 12:50 PM
    0 responses
    49 views
    0 reactions
    Last Post seqadmin  
    Started by seqadmin, 03-03-2025, 01:15 PM
    0 responses
    200 views
    0 reactions
    Last Post seqadmin  
    Working...