Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • Illumina sequencing Quality error

    Hi, everyone!
    Recently, i received the RNA-seq data, when i use FASTX_TOOKIT preprocessing the data, it shows that my data invalid, quality < 0; Is
    there anyone encountered this problem before? thank you!

  • #2
    Sounds to me as if FastX is expecting different quality encoding than you've got in your samples. Which platform does your data come from? What did you want to do with the FastX Toolkit? (I must admit I am not too experienced with that toolkit)

    Comment


    • #3
      append the '-Q 33' command to fastX tools to allow them to work with quality scores that have a base of 33. This is undocumented in the command-line help for the tools, but should work with all tools that demand particular quality values.

      Comment


      • #4
        my data come from Illumina Hiseq 2000 , FASTX_Toolkit can trim the low quality bases, cut off the low quality bases and mask the reads.

        Comment


        • #5
          Originally posted by gringer View Post
          append the '-Q 33' command to fastX tools to allow them to work with quality scores that have a base of 33. This is undocumented in the command-line help for the tools, but should work with all tools that demand particular quality values.
          Hi, gringer, in my reads , if there have more than 1 bases quality lower than 0 , or so ito large amount of reads, my be we can't know which parameter to append ?

          Comment


          • #6
            Hi, gringer, in my reads , if there have more than 1 bases quality lower than 0 , or so ito large amount of reads, my be we can't know which parameter to append ?
            My experience with fastx toolkit is that it doesn't support quality values lower than 0. If you have any in there, then you have to use -Q 33, and make the adjustment manually.

            Comment


            • #7
              Hi, gringer. I want to know "make the adjustment msnuslly" means? thank you !

              Comment


              • #8
                Here's the table from Wikipedia:
                Code:
                  SSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSSS.....................................................
                  ..........................XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX......................
                  ...............................IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII......................
                  .................................JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJ......................
                  LLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLLL....................................................
                  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~
                  |                         |    |        |                              |                     |
                 33                        59   64       73                            104                   126
                
                 S - Sanger        Phred+33,  raw reads typically (0, 40)
                 X - Solexa        Solexa+64, raw reads typically (-5, 40)
                 I - Illumina 1.3+ Phred+64,  raw reads typically (0, 40)
                 J - Illumina 1.5+ Phred+64,  raw reads typically (3, 40)
                    with 0=unused, 1=unused, 2=Read Segment Quality Control Indicator (bold) 
                    (Note: See discussion above).
                 L - Illumina 1.8+ Phred+33,  raw reads typically (0, 41)
                The only negative reads here are the old Solexa reads. These start at base 59, so you could either use 59 as a base for the fastx-toolkit and add 5 to whatever you use for quality thresholds (e.g. mask at q25, rather than q20), or use 33 as a base for everything and add the difference (e.g. mask at q51, rather than q20). This mental arithmetic is what I mean by doing it manually.

                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
                66 views
                0 likes
                Last Post seqadmin  
                Working...
                X