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  • #16
    Official link for this data at NCBI is: http://www.ncbi.nlm.nih.gov/geo/quer...i?acc=GSE32038 (your link seems to be from Taiwan).

    I don't think GEO dataset has fly genome indexes. Those should be downloaded from Illumina igenomes page here: http://support.illumina.com/sequenci...e/igenome.html (See link for BDGP5.25).

    Comment


    • #17
      and of these:
      Drosophila melanogaster Ensembl BDGP5 BDGP5.25
      NCBI build5.41 build5.3 build5 build4.1

      which one should I download?
      You have been my informatic super hero
      Last edited by lupid; 03-20-2015, 09:18 AM.

      Comment


      • #18
        Get Ensembl BDGP5.25 since that is used in Nature Protocols. (any of them would work).

        Comment


        • #19
          You are the best, thank, that work

          Comment


          • #20
            Now the error is this
            Building Bowtie index from GSM794483_C1_R1.transcripts.fa
            [FAILED]
            Error: Couldn't build bowtie index with err = 1

            I tried to put the fata file in the directory, and not change, what can I do now?

            Comment


            • #21
              Use this file instead of the GTF file you are using: /path_to/Drosophila_melanogaster/Ensembl/BDGP5.25/Annotation/Genes/genes.gtf

              Comment


              • #22
                don't work u.u
                tophat -p 2 -G ./genes.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq

                [2015-03-20 16:13:45] Beginning TopHat run (v2.0.13)
                -----------------------------------------------
                [2015-03-20 16:13:45] Checking for Bowtie
                Bowtie version: 2.2.5.0
                [2015-03-20 16:13:45] Checking for Bowtie index files (genome)..
                [2015-03-20 16:13:45] Checking for reference FASTA file
                Warning: Could not find FASTA file ./genome.fa
                [2015-03-20 16:13:45] Reconstituting reference FASTA file from Bowtie index
                Executing: /home/tomas/bin/bowtie2-inspect ./genome > ./C1_R1_thout_new/tmp/genome.fa
                [2015-03-20 16:13:54] Generating SAM header for ./genome
                [2015-03-20 16:13:55] Reading known junctions from GTF file
                [2015-03-20 16:13:59] Preparing reads
                left reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
                right reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
                [2015-03-20 16:18:58] Building transcriptome data files ./C1_R1_thout_new/tmp/genes
                [2015-03-20 16:19:07] Building Bowtie index from genes.fa
                [FAILED]
                Error: Couldn't build bowtie index with err = 1
                tomas@HP-Pavilion:~/bin/c1$

                Comment


                • #23
                  Can you show a current long listing of the files in directory?

                  Code:
                  $ ls -lh
                  As shown on page 570 of Nature protocols article it may be best to create at least this softlink in your current directory. I am assuming you have copied other files locally (will know from the listing above).

                  Code:
                  $ ln -s /path_to/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa .
                  Last edited by GenoMax; 03-20-2015, 12:06 PM.

                  Comment


                  • #24
                    tomas@HP-Pavilion:~/bin/c1$ ls -lh
                    total 4,4G
                    drwxrwxr-x 4 tomas tomas 4,0K mar 20 10:32 C1_R1_thout
                    drwxrwxr-x 4 tomas tomas 4,0K mar 20 14:57 C1_R1_thout_new
                    drwxrwxr-x 3 tomas tomas 4,0K mar 20 15:54 Drosophila_melanogaster
                    -rwxrwxr-x 1 tomas tomas 117M mar 15 2012 genes.fa
                    -rwxrwxr-x 1 tomas tomas 54M may 23 2014 genes.gtf
                    -rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.1.bt2
                    -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.2.bt2
                    -rwxrwxr-x 1 tomas tomas 143 abr 10 2012 genome.3.bt2
                    -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.4.bt2
                    lrwxrwxrwx 1 tomas tomas 29 mar 20 16:51 genome.fa -> ../WholeGenomeFasta/genome.fa
                    -rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.rev.1.bt2
                    -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.rev.2.bt2
                    -rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:34 GSM794483_C1_R1_1.fq
                    -rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:35 GSM794483_C1_R1_2.fq
                    -rw-rw-r-- 1 tomas tomas 138M sep 13 2011 GSM794483_C1_R1_2.fq.gz
                    -rw-rw-r-- 1 tomas tomas 325M sep 9 2011 GSM794483_C1_R1.accepted_hits.bam
                    -rw-rw-r-- 1 tomas tomas 14M mar 20 11:02 GSM794483_C1_R1.transcripts.gff
                    tomas@HP-Pavilion:~/bin/c1$


                    I don't know if I did what you propouse, but when I try, this happend
                    tomas@HP-Pavilion:~/bin/c1$ ln -s /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa .
                    ln: fallo al crear el enlace simbólico «./genome.fa»: El archivo ya existe
                    tomas@HP-Pavilion:~/bin/c1$

                    Comment


                    • #25
                      This link is not pointing to the right directory:

                      lrwxrwxrwx 1 tomas tomas 29 mar 20 16:51 genome.fa -> ../WholeGenomeFasta/genome.fa

                      So do this

                      Code:
                      $ unlink genome.fa
                      ONLY if above does not work then

                      Code:
                      $ rm -f genome.fa
                      Then create the soft link again

                      Code:
                      $ ln -s /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa .
                      Try tophat.

                      Comment


                      • #26
                        after try both options still didn't work
                        here my console
                        tomas@HP-Pavilion:~/bin/c1$ rm -f genome.fa
                        tomas@HP-Pavilion:~/bin/c1$ ln -s /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa .
                        tomas@HP-Pavilion:~/bin/c1$ ls -lh
                        total 4,4G
                        drwxrwxr-x 4 tomas tomas 4,0K mar 20 10:32 C1_R1_thout
                        drwxrwxr-x 4 tomas tomas 4,0K mar 20 14:57 C1_R1_thout_new
                        drwxrwxr-x 3 tomas tomas 4,0K mar 20 15:54 Drosophila_melanogaster
                        -rwxrwxr-x 1 tomas tomas 117M mar 15 2012 genes.fa
                        -rwxrwxr-x 1 tomas tomas 54M may 23 2014 genes.gtf
                        -rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.1.bt2
                        -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.2.bt2
                        -rwxrwxr-x 1 tomas tomas 143 abr 10 2012 genome.3.bt2
                        -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.4.bt2
                        lrwxrwxrwx 1 tomas tomas 91 mar 20 17:23 genome.fa -> /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa
                        -rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.rev.1.bt2
                        -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.rev.2.bt2
                        -rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:34 GSM794483_C1_R1_1.fq
                        -rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:35 GSM794483_C1_R1_2.fq
                        -rw-rw-r-- 1 tomas tomas 138M sep 13 2011 GSM794483_C1_R1_2.fq.gz
                        -rw-rw-r-- 1 tomas tomas 325M sep 9 2011 GSM794483_C1_R1.accepted_hits.bam
                        -rw-rw-r-- 1 tomas tomas 14M mar 20 11:02 GSM794483_C1_R1.transcripts.gff
                        tomas@HP-Pavilion:~/bin/c1$ tophat -p 2 -G ./genes.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq

                        [2015-03-20 17:24:20] Beginning TopHat run (v2.0.13)
                        -----------------------------------------------
                        [2015-03-20 17:24:20] Checking for Bowtie
                        Bowtie version: 2.2.5.0
                        [2015-03-20 17:24:20] Checking for Bowtie index files (genome)..
                        [2015-03-20 17:24:20] Checking for reference FASTA file
                        [2015-03-20 17:24:20] Generating SAM header for ./genome
                        [2015-03-20 17:24:21] Reading known junctions from GTF file
                        [2015-03-20 17:24:24] Preparing reads
                        left reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
                        right reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
                        [2015-03-20 17:29:19] Building transcriptome data files ./C1_R1_thout_new/tmp/genes
                        [2015-03-20 17:29:26] Building Bowtie index from genes.fa
                        [FAILED]
                        Error: Couldn't build bowtie index with err = 1
                        tomas@HP-Pavilion:~/bin/c1$

                        Comment


                        • #27
                          Do this

                          Code:
                          $ head -5 genes.fa
                          and then move the genes.fa file to something else? I am not sure where that file came from

                          Code:
                          $ mv genes.fa genes.fa.BKP

                          Comment


                          • #28
                            Don't u.u
                            I changed the name of the gtf file, because I read that maybe has to have the same as the rest of the files, but don't work either, here my console
                            /bin/c1$ tophat -p 2 -G ./genome.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq

                            [2015-03-20 19:36:20] Beginning TopHat run (v2.0.13)
                            -----------------------------------------------
                            [2015-03-20 19:36:20] Checking for Bowtie
                            Bowtie version: 2.2.5.0
                            [2015-03-20 19:36:20] Checking for Bowtie index files (genome)..
                            [2015-03-20 19:36:20] Checking for reference FASTA file
                            [2015-03-20 19:36:20] Generating SAM header for ./genome
                            [2015-03-20 19:36:20] Reading known junctions from GTF file
                            [2015-03-20 19:36:24] Preparing reads
                            left reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
                            right reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
                            [2015-03-20 19:41:34] Building transcriptome data files ./C1_R1_thout_new/tmp/genome
                            [2015-03-20 19:41:42] Building Bowtie index from genome.fa
                            [FAILED]
                            Error: Couldn't build bowtie index with err = 1
                            tomas@HP-Pavilion:~/bin/c1$ ls -lh
                            total 4,3G
                            -rw-rw-r-- 1 tomas tomas 22K mar 20 19:27 20.3.15
                            -rw-rw-r-- 1 tomas tomas 22K mar 20 19:27 20.3.15.txt
                            drwxrwxr-x 4 tomas tomas 4,0K mar 20 10:32 C1_R1_thout
                            drwxrwxr-x 4 tomas tomas 4,0K mar 20 14:57 C1_R1_thout_new
                            drwxrwxr-x 3 tomas tomas 4,0K mar 20 15:54 Drosophila_melanogaster
                            -rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.1.bt2
                            -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.2.bt2
                            -rwxrwxr-x 1 tomas tomas 143 abr 10 2012 genome.3.bt2
                            -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.4.bt2
                            lrwxrwxrwx 1 tomas tomas 91 mar 20 17:23 genome.fa -> /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/Bowtie2Index/genome.fa
                            -rwxrwxr-x 1 tomas tomas 54M may 23 2014 genome.gtf
                            -rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.rev.1.bt2
                            -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.rev.2.bt2
                            -rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:34 GSM794483_C1_R1_1.fq
                            -rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:35 GSM794483_C1_R1_2.fq
                            -rw-rw-r-- 1 tomas tomas 138M sep 13 2011 GSM794483_C1_R1_2.fq.gz
                            -rw-rw-r-- 1 tomas tomas 325M sep 9 2011 GSM794483_C1_R1.accepted_hits.bam
                            -rw-rw-r-- 1 tomas tomas 14M mar 20 11:02 GSM794483_C1_R1.transcripts.gff

                            Comment


                            • #29
                              Did you download bowtie2 and tophat executables or compiled them yourself? Are you running 32-bit/64-bit Ubuntu?

                              Since you have copied all other files to the current directory why don't we copy the genome file too
                              Code:
                              $ unlink genome.fa
                              $ cp /home/tomas/bin/c1/Drosophila_melanogaster/Ensembl/BDGP5.25/Sequence/WholeGenomeFasta/genome.fa .

                              Comment


                              • #30
                                I'm using Ubuntu 64 and download the executables
                                I try to put the file in the same directory as you suggest and nothing
                                /bin/c1$ ls -lh
                                total 4,4G
                                -rw-rw-r-- 1 tomas tomas 22K mar 20 19:27 20.3.15
                                -rw-rw-r-- 1 tomas tomas 22K mar 20 19:27 20.3.15.txt
                                drwxrwxr-x 4 tomas tomas 4,0K mar 20 10:32 C1_R1_thout
                                drwxrwxr-x 4 tomas tomas 4,0K mar 20 14:57 C1_R1_thout_new
                                drwxrwxr-x 3 tomas tomas 4,0K mar 20 15:54 Drosophila_melanogaster
                                -rwxrwxr-x 1 tomas tomas 54M mar 20 20:02 genes.gtf
                                -rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.1.bt2
                                -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.2.bt2
                                -rwxrwxr-x 1 tomas tomas 143 abr 10 2012 genome.3.bt2
                                -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.4.bt2
                                -rwxrwxr-x 1 tomas tomas 117M mar 21 10:16 genome.fa
                                -rwxrwxr-x 1 tomas tomas 43M abr 10 2012 genome.rev.1.bt2
                                -rwxrwxr-x 1 tomas tomas 29M abr 10 2012 genome.rev.2.bt2
                                -rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:34 GSM794483_C1_R1_1.fq
                                -rw-rw-r-- 1 tomas tomas 1,8G mar 20 10:35 GSM794483_C1_R1_2.fq
                                -rw-rw-r-- 1 tomas tomas 138M sep 13 2011 GSM794483_C1_R1_2.fq.gz
                                -rw-rw-r-- 1 tomas tomas 325M sep 9 2011 GSM794483_C1_R1.accepted_hits.bam
                                -rw-rw-r-- 1 tomas tomas 14M mar 20 11:02 GSM794483_C1_R1.transcripts.gff
                                tomas@HP-Pavilion:~/bin/c1$ tophat -p 2 -G ./genes.gtf -o ./C1_R1_thout_new ./genome ./GSM794483_C1_R1_1.fq ./GSM794483_C1_R1_2.fq

                                [2015-03-21 10:16:47] Beginning TopHat run (v2.0.13)
                                -----------------------------------------------
                                [2015-03-21 10:16:47] Checking for Bowtie
                                Bowtie version: 2.2.5.0
                                [2015-03-21 10:16:48] Checking for Bowtie index files (genome)..
                                [2015-03-21 10:16:48] Checking for reference FASTA file
                                [2015-03-21 10:16:48] Generating SAM header for ./genome
                                [2015-03-21 10:16:50] Reading known junctions from GTF file
                                [2015-03-21 10:16:54] Preparing reads
                                left reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
                                right reads: min. length=75, max. length=75, 11607353 kept reads (0 discarded)
                                [2015-03-21 10:22:22] Building transcriptome data files ./C1_R1_thout_new/tmp/genes
                                [2015-03-21 10:22:29] Building Bowtie index from genes.fa
                                [FAILED]
                                Error: Couldn't build bowtie index with err = 1
                                tomas@HP-Pavilion:~/bin/c1$

                                Comment

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