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  • mapardo
    Junior Member
    • Jun 2011
    • 8
    #1

    incoherent result cufflinks

    I have done a easy test with cufflinks and the result is not coherent with the input files.

    I use strand specific data (input.sam).

    ILLUMINA+GA_0000:1:1:1079:18161#0 99 chr3 1355 255 36M = 1366 47 TTCAGGAGGCCACCCGACATAGTGCAGAGAAGCGGA YccacSccc\ccccXcYScc]a]a\caScc^acccK NM:i:0 NH:i:1 XS:A:-
    ILLUMINA+GA_0000:1:1:1079:18161#0 147 chr3 1366 255 36M = 1355 +47 ACCCGACATANTNCAGAGAAGCGGCCCACAATGCGN BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB NM:i:4 NH:i:1 XS:A:-
    ILLUMINA+GA_0000:1:1:1079:18161#0 99 chr3 1355 255 36M = 1366 47 TTCAGGAGGCCACCCGACATAGTGCAGAGAAGCGGA YccacSccc\ccccXcYScc]a]a\caScc^acccK NM:i:0 NH:i:1 XS:A:-
    ILLUMINA+GA_0000:1:1:1079:18161#0 147 chr3 1366 255 36M = 1355 +47 ACCCGACATANTNCAGAGAAGCGGCCCACAATGCGN BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB NM:i:4 NH:i:1 XS:A:-
    ILLUMINA+GA_0000:1:1:1079:18161#0 99 chr3 1355 255 36M = 1366 47 TTCAGGAGGCCACCCGACATAGTGCAGAGAAGCGGA YccacSccc\ccccXcYScc]a]a\caScc^acccK NM:i:0 NH:i:1 XS:A:-
    ILLUMINA+GA_0000:1:1:1079:18161#0 147 chr3 1366 255 36M = 1355 +47 ACCCGACATANTNCAGAGAAGCGGCCCACAATGCGN BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB NM:i:4 NH:i:1 XS:A:-
    ILLUMINA-GA_0000:1:1:1080:14412#0 83 chr3 9861 255 36M = 9828 -69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA c]\b]JccYcccccPcaRb]]Jb]aZYYaccYaccc NM:i:0 NH:i:1 XS:A:+
    ILLUMINA-GA_0000:1:1:1080:14412#0 163 chr3 9828 255 36M = 9861 69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB NM:i:3 NH:i:1 XS:A:+
    ILLUMINA-GA_0000:1:1:1080:14412#0 99 chr3 9828 255 36M = 9861 69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA c]\b]JccYcccccPcaRb]]Jb]aZYYaccYaccc NM:i:0 NH:i:1 XS:A:-
    ILLUMINA-GA_0000:1:1:1080:14412#0 147 chr3 9861 255 36M = 9828 +69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB NM:i:3 NH:i:1 XS:A:-
    ILLUMINA-GA_0000:1:1:1080:14412#0 99 chr3 9828 255 36M = 9861 69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA c]\b]JccYcccccPcaRb]]Jb]aZYYaccYaccc NM:i:0 NH:i:1 XS:A:-
    ILLUMINA-GA_0000:1:1:1080:14412#0 147 chr3 9861 255 36M = 9828 +69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB NM:i:3 NH:i:1 XS:A:-
    ILLUMINA-GA_0000:1:1:1080:14412#0 99 chr3 9828 255 36M = 9861 69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA c]\b]JccYcccccPcaRb]]Jb]aZYYaccYaccc NM:i:0 NH:i:1 XS:A:-
    ILLUMINA-GA_0000:1:1:1080:14412#0 147 chr3 9861 255 36M = 9828 +69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB NM:i:3 NH:i:1 XS:A:-


    I sort the file with samtools (input.sort.bam) and use this command for Cufflinks:

    cufflinks --library-type fr-firststrand --min-frags-per-transfrag 3 input.sort.bam

    The result is the file transcript3.gtf, where transcripts have no sense.

    chr3 Cufflinks transcript 1355 1401 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; FPKM "8019117703.6926527023"; frac "1.000000"; conf_lo "0.000000"; conf_hi "17278797233.473148"; cov "4041.635323";
    chr3 Cufflinks exon 1355 1401 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; exon_number "1"; FPKM "8019117703.6926527023"; frac "1.000000"; conf_lo "0.000000"; conf_hi "17278797233.473148"; cov "4041.635323";
    chr3 Cufflinks transcript 9828 9896 1000 + . gene_id "CUFF.2"; transcript_id "CUFF.2.1"; FPKM "1157774097.4185056686"; frac "1.000000"; conf_lo "0.000000"; conf_hi "2315548194.837011"; cov "583.518145";
    chr3 Cufflinks exon 9828 9896 1000 + . gene_id "CUFF.2"; transcript_id "CUFF.2.1"; exon_number "1"; FPKM "1157774097.4185056686"; frac "1.000000"; conf_lo "0.000000"; conf_hi "2315548194.837011"; cov "583.518145";


    If I use a different value for --min-frags-per-transfrag parameter, results change to the correct one

    cufflinks --library-type fr-firststrand --min-frags-per-transfrag 1 input.sort.bam

    File transcript1.gtf.


    chr3 Cufflinks transcript 1355 1401 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; FPKM "8019117703.6926527023"; frac "1.000000"; conf_lo "0.000000"; conf_hi "17278797233.473148"; cov "4041.635323";
    chr3 Cufflinks exon 1355 1401 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; exon_number "1"; FPKM "8019117703.6926527023"; frac "1.000000"; conf_lo "0.000000"; conf_hi "17278797233.473148"; cov "4041.635323";
    chr3 Cufflinks transcript 9828 9896 1000 + . gene_id "CUFF.2"; transcript_id "CUFF.2.1"; FPKM "289443524.3546264172"; frac "0.250000"; conf_lo "0.000000"; conf_hi "868330573.063879"; cov "145.879536";
    chr3 Cufflinks exon 9828 9896 1000 + . gene_id "CUFF.2"; transcript_id "CUFF.2.1"; exon_number "1"; FPKM "289443524.3546264172"; frac "0.250000"; conf_lo "0.000000"; conf_hi "868330573.063879"; cov "145.879536";
    chr3 Cufflinks transcript 9828 9896 1000 - . gene_id "CUFF.3"; transcript_id "CUFF.3.1"; FPKM "868330573.0638793707"; frac "0.750000"; conf_lo "0.000000"; conf_hi "1870992353.271905"; cov "437.638609";
    chr3 Cufflinks exon 9828 9896 1000 - . gene_id "CUFF.3"; transcript_id "CUFF.3.1"; exon_number "1"; FPKM "868330573.0638793707"; frac "0.750000"; conf_lo "0.000000"; conf_hi "1870992353.271905"; cov "437.638609";


    There is something I have missed?
    Tags: cufflinks, error, result
    • swbarnes2
      Senior Member
      • May 2008
      • 910
      #2
      First thing anyone thinks when seeing a string of Bs for a quality string is that you are using an oldish dataset, and those reads are the worst possible quailty. You appear to be using phred 64 offset quality scaling. So a 'B' is a 66 in ASCII, which makes it a 2 in quality. An 'h' on the other hand, is a 104 in ASCII, which is a quality of 40.

      So try your test on higher quality reads for starters. If the quality string has a lot of h's that's a good quality read.

      Newer runs have phred offsets of 33, so that '#' is the lowest quality, and 'I' is the highest.

        Comment

        • mapardo
          Junior Member
          • Jun 2011
          • 8
          #3
          Thanks for the quick reply.

          I have changed the quality of the reads to 'h' for all the nt but the result is the same.

          INPUT FILE 1
          ILLUMINA+GA_0000:1:1:1079:18161#0 99 chr3 1355 255 36M = 1366 47 TTCAGGAGGCCACCCGACATAGTGCAGAGAAGCGGA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18161#0 147 chr3 1366 255 36M = 1355 +47 ACCCGACATANTNCAGAGAAGCGGCCCACAATGCGN hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18162#0 99 chr3 1355 255 36M = 1366 47 TTCAGGAGGCCACCCGACATAGTGCAGAGAAGCGGA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18162#0 147 chr3 1366 255 36M = 1355 +47 ACCCGACATANTNCAGAGAAGCGGCCCACAATGCGN hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18163#0 99 chr3 1355 255 36M = 1366 47 TTCAGGAGGCCACCCGACATAGTGCAGAGAAGCGGA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18163#0 147 chr3 1366 255 36M = 1355 +47 ACCCGACATANTNCAGAGAAGCGGCCCACAATGCGN hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14414#0 83 chr3 9861 255 36M = 9828 -69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:+
          ILLUMINA+GA_0000:1:1:1080:14414#0 163 chr3 9828 255 36M = 9861 69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:+
          ILLUMINA+GA_0000:1:1:1080:14415#0 99 chr3 9828 255 36M = 9861 69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14415#0 147 chr3 9861 255 36M = 9828 +69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14416#0 99 chr3 9828 255 36M = 9861 69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14416#0 147 chr3 9861 255 36M = 9828 +69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14417#0 99 chr3 9828 255 36M = 9861 69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14417#0 147 chr3 9861 255 36M = 9828 +69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-


          RESULT 1
          chr3 Cufflinks transcript 1355 1401 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; FPKM "8019117703.6926527023"; frac "1.000000"; conf_lo "0.000000"; conf_hi "17278797233.473148"; cov "4041.635323";
          chr3 Cufflinks exon 1355 1401 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; exon_number "1"; FPKM "8019117703.6926527023"; frac "1.000000"; conf_lo "0.000000"; conf_hi "17278797233.473148"; cov "4041.635323";
          chr3 Cufflinks transcript 9828 9896 1000 + . gene_id "CUFF.2"; transcript_id "CUFF.2.1"; FPKM "1157774097.4185056686"; frac "1.000000"; conf_lo "0.000000"; conf_hi "2315548194.837011"; cov "583.518145";
          chr3 Cufflinks exon 9828 9896 1000 + . gene_id "CUFF.2"; transcript_id "CUFF.2.1"; exon_number "1"; FPKM "1157774097.4185056686"; frac "1.000000"; conf_lo "0.000000"; conf_hi "2315548194.837011"; cov "583.518145";




          If I change the position of the less abundant fragment, the result is the correct.

          INPUT FILE 2
          ILLUMINA+GA_0000:1:1:1079:18161#0 99 chr3 1355 255 36M = 1366 47 TTCAGGAGGCCACCCGACATAGTGCAGAGAAGCGGA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18161#0 147 chr3 1366 255 36M = 1355 +47 ACCCGACATANTNCAGAGAAGCGGCCCACAATGCGN hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18162#0 99 chr3 1355 255 36M = 1366 47 TTCAGGAGGCCACCCGACATAGTGCAGAGAAGCGGA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18162#0 147 chr3 1366 255 36M = 1355 +47 ACCCGACATANTNCAGAGAAGCGGCCCACAATGCGN hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18163#0 99 chr3 1355 255 36M = 1366 47 TTCAGGAGGCCACCCGACATAGTGCAGAGAAGCGGA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1079:18163#0 147 chr3 1366 255 36M = 1355 +47 ACCCGACATANTNCAGAGAAGCGGCCCACAATGCGN hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14414#0 83 chr3 2861 255 36M = 9828 -69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:+
          ILLUMINA+GA_0000:1:1:1080:14414#0 163 chr3 2828 255 36M = 9861 69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:+
          ILLUMINA+GA_0000:1:1:1080:14415#0 99 chr3 9828 255 36M = 9861 69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14415#0 147 chr3 9861 255 36M = 9828 +69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14416#0 99 chr3 9828 255 36M = 9861 69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14416#0 147 chr3 9861 255 36M = 9828 +69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14417#0 99 chr3 9828 255 36M = 9861 69 CTTCATCCGCCAACTAATATTTCACTTTACATCCAA hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-
          ILLUMINA+GA_0000:1:1:1080:14417#0 147 chr3 9861 255 36M = 9828 +69 NGTCATTATTGGCTCAACTTTCCNCNCTATCTGCTT hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh NM:i:0 NH:i:1 XS:A:-

          RESULT 2
          chr3 Cufflinks transcript 1355 1401 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; FPKM "8019117703.6926527023"; frac "1.000000"; conf_lo "0.000000"; conf_hi "17278797233.473148"; cov "4041.635323";
          chr3 Cufflinks exon 1355 1401 1000 - . gene_id "CUFF.1"; transcript_id "CUFF.1.1"; exon_number "1"; FPKM "8019117703.6926527023"; frac "1.000000"; conf_lo "0.000000"; conf_hi "17278797233.473148"; cov "4041.635323";
          chr3 Cufflinks transcript 9828 9896 1000 - . gene_id "CUFF.2"; transcript_id "CUFF.2.1"; FPKM "868330573.0638793707"; frac "1.000000"; conf_lo "0.000000"; conf_hi "1870992353.271905"; cov "437.638609";
          chr3 Cufflinks exon 9828 9896 1000 - . gene_id "CUFF.2"; transcript_id "CUFF.2.1"; exon_number "1"; FPKM "868330573.0638793707"; frac "1.000000"; conf_lo "0.000000"; conf_hi "1870992353.271905"; cov "437.638609";

          In both runs I have been using the same parameters for cufflinks:

          cufflinks --library-type fr-firststrand -F 0.01 --min-frags-per-transfrag 2 input.sort.bam

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