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  • cuffdiff for groups

    I want run cuffdiff for two groups. each has 3 samples. Is the below code right?

    cuffdiff -o diff_out -b ../genome/ce10.fa -p 2 -L em,la -u merged_asm/merged.gtf cond1.bam1,cond1.bam2,cond1.bam3 cond1.bam1,cond1.bam2,cond1.bam3
    Last edited by bvk; 05-05-2015, 05:33 AM.

  • #2
    No. That is not correct.

    -L has to be a comma separated list of condition labels.

    -L cond1,cond2 (e.g. -L Control,Mutant)

    Your list of bam files (comma delimited names no spaces, then two conditions separated by a space) will be. e.g. cond1.bam1,cond1.bam2,cond1.bam3 (space here) cond2.bam1,cond2.bam2,cond2.bam3
    Last edited by GenoMax; 05-05-2015, 03:16 AM.

    Comment


    • #3
      Originally posted by GenoMax View Post
      No. That is not correct.

      -L has to be a comma separated list of condition labels.

      -L cond1,cond2 (e.g. -L Control,Mutant)

      Your list of bam files (comma delimited names no spaces, then two conditions separated by a space) will be. e.g. cond1.bam1,cond1.bam2,cond1.bam3 (space here) cond2.bam1,cond2.bam2,cond2.bam3
      You missed it. please, see it after 3rd bam file I haven't given any comma. And it is separated with space too.

      Comment


      • #4
        The -L option you have used in your command is not two group names for three samples in each group.

        ", ../tophat/la/SRR493365_thout/accepted_hits.bam " It also looks like there is a space between , and the .. but that may be an illusion in the way the browser is displaying it.

        Comment


        • #5
          Aside from your syntax problems, you have a more serious issue in that you're not specifying the actual experiment correctly. SRR493359, SRR493360 and SRR493361 are from the same sample and should just be merged together into a single BAM file. Similarly SRR493363, SRR493364 and SRR493365 are from the same sample. So, you actually have a 1 vs. 1 sample comparison. Do NOT lump each of the files for each sample into a group, since then you're making fake replicates and will have largely meaningless results (of course, you're doing a 1 vs. 1 comparison, so the results aren't exactly robust to begin with).

          Comment


          • #6
            Originally posted by GenoMax View Post
            The -L option you have used in your command is not two group names for three samples in each group.

            ", ../tophat/la/SRR493365_thout/accepted_hits.bam " It also looks like there is a space between , and the .. but that may be an illusion in the way the browser is displaying it.
            ok. now the below code looks fine I guess

            cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L SRR493359,SRR493360,SRR493361,SRR493363,SRR493364,SRR493365 -u merged_asm/merged.gtf ../tophat/em/SRR493359_thout/accepted_hits.bam,../tophat/em/SRR493360_thout/accepted_hits.bam,../tophat/em/SRR493361_thout/accepted_hits.bam ../tophat/la/SRR493363_thout/accepted_hits.bam,../tophat/la/SRR493364_thout/accepted_hits.bam,../tophat/la/SRR493365_thout/accepted_hits.bam

            Comment


            • #7
              Originally posted by bvk View Post
              ok. now the below code looks fine I guess

              cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L SRR493359,SRR493360,SRR493361,SRR493363,SRR493364,SRR493365 -u merged_asm/merged.gtf ../tophat/em/SRR493359_thout/accepted_hits.bam,../tophat/em/SRR493360_thout/accepted_hits.bam,../tophat/em/SRR493361_thout/accepted_hits.bam ../tophat/la/SRR493363_thout/accepted_hits.bam,../tophat/la/SRR493364_thout/accepted_hits.bam,../tophat/la/SRR493365_thout/accepted_hits.bam
              No, it's not. You're making two groups with the BAM files and are giving those two groups 6 labels rather than 2. "-L larval,early" would make more sense, though see my earlier reply.

              Comment


              • #8
                Originally posted by dpryan View Post
                Aside from your syntax problems, you have a more serious issue in that you're not specifying the actual experiment correctly. SRR493359, SRR493360 and SRR493361 are from the same sample and should just be merged together into a single BAM file. Similarly SRR493363, SRR493364 and SRR493365 are from the same sample. So, you actually have a 1 vs. 1 sample comparison. Do NOT lump each of the files for each sample into a group, since then you're making fake replicates and will have largely meaningless results (of course, you're doing a 1 vs. 1 comparison, so the results aren't exactly robust to begin with).
                so you say that it should look like this:

                cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L SRR493359,SRR493360,SRR493361,SRR493363,SRR493364,SRR493365 -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

                SRR493359_60_61_thout which has merged bam file of 59,60 and 61
                SRR493363_64_65_thout which has merged bam file of 63,64 and 65

                Comment


                • #9
                  Originally posted by bvk View Post
                  so you say that it should look like this:

                  cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L SRR493359,SRR493360,SRR493361,SRR493363,SRR493364,SRR493365 -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

                  SRR493359_60_61_thout which has merged bam file of 59,60 and 61
                  SRR493363_64_65_thout which has merged bam file of 63,64 and 65
                  Arrange the labels (larval/early) according to their correspondence with the BAM files.

                  Code:
                  cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

                  Comment


                  • #10
                    Originally posted by GenoMax View Post
                    Arrange the labels (larval/early) according to their correspondence with the BAM files.

                    Code:
                    cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam
                    Yes, I understood. Thank you very much !!

                    Comment


                    • #11
                      Originally posted by dpryan View Post
                      No, it's not. You're making two groups with the BAM files and are giving those two groups 6 labels rather than 2. "-L larval,early" would make more sense, though see my earlier reply.
                      Thankyou !! I got it.

                      Comment


                      • #12
                        Originally posted by dpryan View Post
                        No, it's not. You're making two groups with the BAM files and are giving those two groups 6 labels rather than 2. "-L larval,early" would make more sense, though see my earlier reply.
                        As you said If I run the following command

                        cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

                        Now, If I want to find the expression levels for each sample I guess it is not possible, cz of the merged bam file.

                        Is it possible to give labels as larval,early and giving bam files of 6 samples. Will this work?

                        for eg:

                        cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L embryo,larva -u merged_asm/merged.gtf ../tophat/em/SRR493359_thout/accepted_hits.bam,../tophat/em/SRR493360_thout/accepted_hits.bam,../tophat/em/SRR493361_thout/accepted_hits.bam ../tophat/la/SRR493363_thout/accepted_hits.bam,../tophat/la/SRR493364_thout/accepted_hits.bam,../tophat/la/SRR493365_thout/accepted_hits.bam

                        Comment


                        • #13
                          You only have 2 samples anyway. Any sort of metric you'd get from each of the subfiles isn't terribly meaningful unless you're interested in looking at technical variance.

                          Comment


                          • #14
                            Originally posted by GenoMax View Post
                            Arrange the labels (larval/early) according to their correspondence with the BAM files.

                            Code:
                            cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam
                            As you said If I run the following command

                            cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L larval,early -u merged_asm/merged.gtf ../tophat/em/SRR493359_60_61_thout/accepted_hits.bam ../tophat/em/SRR493363_64_65_thout/accepted_hits.bam

                            Now, If I want to find the expression levels for each sample I guess it is not possible, cz of the merged bam file.

                            Is it possible to give labels as larval,early and giving bam files of 6 samples. Will this work?

                            for eg:

                            cuffdiff -o diff_out4 -b ../genome/ce10.fa -p 2 -L embryo,larva -u merged_asm/merged.gtf ../tophat/em/SRR493359_thout/accepted_hits.bam,../tophat/em/SRR493360_thout/accepted_hits.bam,../tophat/em/SRR493361_thout/accepted_hits.bam ../tophat/la/SRR493363_thout/accepted_hits.bam,../tophat/la/SRR493364_thout/accepted_hits.bam,../tophat/la/SRR493365_thout/accepted_hits.bam

                            Comment


                            • #15
                              Technically cuffdiff command as outlined above will work. But Devon has already warned you about the consequence in post #5.

                              Comment

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