Seqanswers Leaderboard Ad

Collapse

Announcement

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

  • For paired end libraries, how do I know which is forward and which is reverse?

    For paired end libraries, how do I know which is forward and which is reverse?

    I've got the files in galaxy, but I don't know which are forward and which are reverse, just that they're paired.

  • #2
    File with R1 in the name is the first read and R2 is the second read (other end) from the same set of fragments.

    Comment


    • #3
      The file names don't have those. After the genotype, they just say lane_4 of lane_5

      Comment


      • #4
        Can you post a complete filename, and also the first 8 lines from that file?

        Comment


        • #5
          Have the reads been processed in any way (i.e. collapsed into a single long read, possible if the insert is short and reads overlap)?

          If reads ARE really paired, then they can't be in separate lanes (there would be two files per lane).

          Comment


          • #6
            File name:

            Galaxy3-[WT_2_lane4.fastq].fastq (then the next file is the same, except lane5. each genotype has two files, one lane four and one lane 5)

            @GWZHISEQ02:321YMKACXX:5:1101:1297:1992 1:N:0:ATCACG
            TTTGGATCGTTCACAAATTGAGAGAGGTTTTCGCGTAGGAAGTACTTGNCC
            +
            CCCFFFFFHHHFHJJIIJJJJJJJJJJ@GHJIIJJJGIJEIJBFHIJI###
            @GWZHISEQ02:321YMKACXX:5:1101:1692:1983 1:N:0:ATCACG
            CCCGCCCTTCTTGCCGCGCGGCNGCATCATTCGGTTGGGCACGCCGCNNNN
            +
            @@@DDADDHFFHHIII@GGIIG#07<FDHIIG@F5AGHFEBE??A@B####
            @GWZHISEQ02:321YMKACXX:5:1101:2408:1992 1:N:0:ATCACG
            GTACTTGTAGAGCTCGGTGTATGTAATCCTGTCCTCAGCGGTCCAGCTNAA
            +
            @<<DDEDDHGFHHIDEHCGIIGIDHHJ@HHIGIAFEIGEEGIIIGHIG###
            @GWZHISEQ02:321YMKACXX:5:1101:3073:1994 1:N:0:ATCACG
            CCGAGTTCTCCTGGTACCAGCAGATCGAGATGGGCTACGACCCCCAGCNGA
            +
            @@CFFDFFHHHHHJGHIJJIIJJJJJJJJIIJJIGIHIIJJJJJJJJJ###
            @GWZHISEQ02:321YMKACXX:5:1101:3748:1991 1:N:0:ATCACG
            GTGCGTTCGCTGGAGAATGTGTGCCGCGACCTGATCAACGGTGCAAAGNAC
            +
            @@@DDDDDAHFDFFHFEFHIIJEGGIEGEHIJGBGIGGIIG?EHHFDF###
            @GWZHISEQ02:321YMKACXX:5:1101:3653:1998 1:N:0:ATCACG
            CTGACTGGCATGATCCAGTCGACGATGATGGCCATGCCCTACATGGACAAA
            +
            CCCFFFFFHHHHHJJJJJJJJJJJHJJJJJJJJJJJJJJIJJJJJJJJJJ#
            @GWZHISEQ02:321YMKACXX:5:1101:5002:1996 1:N:0:ATCACG
            CCTCAGTTCTCCTGGTTCCACCAGATCGAGATGGGCTACGATCCACAANTG
            +
            ???;:=2=A=D<DEBE::+<<F3<33A?1811111)11:6)0000?D####
            @GWZHISEQ02:321YMKACXX:5:1101:5470:1986 1:N:0:ATCACG
            CTGGATATCAATAATGCTCTCCNTAGGGATATTTCCCGCAAATTTGANNNN
            +
            CCCFFFFFHHHHHJJJJJJJJJ#3AGIJJJJJJJJJJJJJJJJJJJJ####



            When I try to take the two with the same geno# and map them as paired end, I get this:
            Left reads:
            Input: 22670964
            Mapped: 21232534 (93.7% of input)
            of these: 1499440 ( 7.1%) have multiple alignments (0 have >20)
            Right reads:
            Input: 22516885
            Mapped: 21088604 (93.7% of input)
            of these: 1487965 ( 7.1%) have multiple alignments (0 have >20)
            93.7% overall read alignment rate.

            Aligned pairs: 19856733
            of these: 99084 ( 0.5%) have multiple alignments
            and: 18762256 (94.5%) are discordant alignments
            4.9% concordant pair alignment rate.
            Last edited by skmotay; 10-09-2014, 10:15 AM.

            Comment


            • #7
              These reads are not paired.

              @GWZHISEQ02:321YMKACXX:5:1101:1297:1992 1:N:0:ATCACG

              That 1 indicates it is read 1. So you get a high discordant rate because you are mapping unrelated single-ended reads as if they were paired.

              Comment


              • #8
                If your description is right then they are not paired end reads. Just a sample run in two lanes.

                For them to be true paired-end reads, each sample will need to have two files (from the same lane) with the naming convention described here: http://en.wikipedia.org/wiki/FASTQ_f...ce_identifiers

                Comment


                • #9
                  Well, that explains...a whole lot. And eases as much frustration!

                  (If only the data generator had left some notes or would return an email...)

                  I thought biological reps were unnecessary for RNA seq?

                  Comment


                  • #10
                    Originally posted by skmotay View Post
                    I thought biological reps were unnecessary for RNA seq?
                    It is true that technical replicates are unnecessary but biological replicates are a must if you hope to get any meaningful results.

                    Comment


                    • #11
                      Yup, sorry I meant technical reps are unnecessary.

                      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