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Guide/tutorial for the analysis of RNA-seq data

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  • #61
    Hi,

    I was wondering if there is a typo in the script here:
    Code:
    awk '$3!="*"' untreated1.fa|wc -l
    wc -l untreated1.fa
    Did you mean to check the sam file? (untreated1.sam)
    How can you see in the fastA file how many reads were aligned?

    Thanks

    Comment


    • #62
      Thanks for the nice tutorial

      I was looking for such a tutorial for some time. I got it now. Thanks a lot!

      Comment


      • #63
        Thanks very much.
        This is the right tutorial for me..

        Comment


        • #64
          Hey everyone,
          I am new to NGS and I was trying the DE analysis of Di Arabidopsis pathogen data as mentioned in the tutorial. I followed every step and in the end I got the count of significant genes to be 169. I wanted to know how do I get the file that describes all the significant genes?. Any help will be much appreciated since I am still in the learning phase.
          Thanks in advance,
          Himanshu.

          Comment


          • #65
            Thank you for the very nice tutorial, and also for guiding me to the 'how-to-wiki'!!

            Comment


            • #66
              Hi,

              I am trying to install miRanalyzer stand alone version. I have built the database and managed to download all required tools and packages. When I tried to run it with the test data provided, I get this message: "Failed to load Main-Class manifest attribute from miRanalyzer.jar
              ". Just wondering what I am doing wrong

              Comment


              • #67
                the seven fasta files by Li et al., 2008

                Can anyone help me in giving a link on the seven fasta files by Li et al 2008 used in the RNA-seq tutorial posted just of recent?

                Comment


                • #68
                  Hi

                  When making Exon Junction libraries, you suggest to use Make Splice Junction Fasta (USeq software package). I've read on their web page that it's depreciated and Make Transcriptome (USeq's package as well) should be used instead.

                  Make Transcriptome gives 2 outputs; transcripts and splices. I guess I shall use only splices file in further step(s). Am I right?

                  Comment


                  • #69
                    Hi,
                    I'm very new to RNA sequencing analysis and I've been using Trapnell et al., 2012 protocol as a guide. I've succesfully downloaded the softwares including the fruitfly genome in "my_rna_seq" directory as advised and I try as much as possible to move files extracted in the Downloads to this directory so that I can have everything in the above mentioned directory.
                    The challenge came when I wanted to align my RNA-seq reads to the reference genome. After the first command, all I got was an error indicating that "Could not find Bowtie index files C1_R1_1.fq.*" I'm faced with this error monster and I just need a clue to find this monster.
                    I have the following files in my folder:
                    bowtie-0.12.8
                    bowtie-0.12.8-linux-x86_64.zip
                    C1_R1_thout
                    C1_R1_thout--library-type=fr-firststrand genome
                    d_melanogaster_fb5_22.1.ebwt
                    d_melanogaster_fb5_22.2.ebwt
                    d_melanogaster_fb5_22.3.ebwt
                    d_melanogaster_fb5_22.4.ebwt
                    d_melanogaster_fb5_22.ebwt.zip
                    d_melanogaster_fb5_22.rev.1.ebwt
                    d_melanogaster_fb5_22.rev.2.ebwt
                    Drosophila_melanogaster
                    Drosophila_melanogaster_Ensembl_BDGP5.25.tar.gz
                    Ensem
                    genes.gtf
                    genome.*.
                    GSE32038_simulated_fastq_files.tar.gz
                    GSM794483_C1_R1_1.fq.gz
                    GSM794483_C1_R1_2.fq.gz
                    GSM794484_C1_R2_1.fq.gz
                    GSM794484_C1_R2_2.fq.gz
                    GSM794485_C1_R3_1.fq.gz
                    GSM794485_C1_R3_2.fq.gz
                    GSM794486_C2_R1_1.fq.gz
                    GSM794486_C2_R1_2.fq.gz
                    GSM794487_C2_R2_1.fq.gz
                    GSM794487_C2_R2_2.fq.gz
                    GSM794488_C2_R3_1.fq.gz
                    GSM794488_C2_R3_2.fq.gz
                    make_d_melanogaster_fb5_22.sh
                    README.txt
                    tophat_out

                    and I used this command:
                    tophat -p 8 -G genes.gtf -o C1_R1_thout--library-type=fr-firststrand\ genome C1_R1_1.fq C1_R1_2.fq

                    Thanks.

                    Comment


                    • #70
                      I am not familiar with tophat, but I have been using bowtie.

                      Before the mapping process of bowtie, it should index the reference genome using the command such as:
                      bowtie-build hg19.fasta hg19
                      then six files (hg19.1.ebwt, hg19.2.ebwt, hg19.3.ebwt, hg19.4.ebwt, hg19.rev.1.ebwt, and hg19.rev.2.ebwt) will be generated.

                      During the process of mapping, bowtie searched the short reads(usually *.fq) again those six indexed files.

                      I think the index process and mapping process have been automated by tophat.

                      In your question, it reports "Couldn't find index files C1_R1_1.fq.* ".
                      I guess the reference genome should be d_melanogaster_fb5_22

                      did you forget to specify the reference genome in your command?
                      tophat -p 8 -G genes.gtf -o C1_R1_thout--library-type=fr-firststrand\ genome C1_R1_1.fq C1_R1_2.fq

                      hope can help you



                      Originally posted by Ajayi Oyeyemi View Post
                      Hi,
                      I'm very new to RNA sequencing analysis and I've been using Trapnell et al., 2012 protocol as a guide. I've succesfully downloaded the softwares including the fruitfly genome in "my_rna_seq" directory as advised and I try as much as possible to move files extracted in the Downloads to this directory so that I can have everything in the above mentioned directory.
                      The challenge came when I wanted to align my RNA-seq reads to the reference genome. After the first command, all I got was an error indicating that "Could not find Bowtie index files C1_R1_1.fq.*" I'm faced with this error monster and I just need a clue to find this monster.
                      I have the following files in my folder:
                      bowtie-0.12.8
                      bowtie-0.12.8-linux-x86_64.zip
                      C1_R1_thout
                      C1_R1_thout--library-type=fr-firststrand genome
                      d_melanogaster_fb5_22.1.ebwt
                      d_melanogaster_fb5_22.2.ebwt
                      d_melanogaster_fb5_22.3.ebwt
                      d_melanogaster_fb5_22.4.ebwt
                      d_melanogaster_fb5_22.ebwt.zip
                      d_melanogaster_fb5_22.rev.1.ebwt
                      d_melanogaster_fb5_22.rev.2.ebwt
                      Drosophila_melanogaster
                      Drosophila_melanogaster_Ensembl_BDGP5.25.tar.gz
                      Ensem
                      genes.gtf
                      genome.*.
                      GSE32038_simulated_fastq_files.tar.gz
                      GSM794483_C1_R1_1.fq.gz
                      GSM794483_C1_R1_2.fq.gz
                      GSM794484_C1_R2_1.fq.gz
                      GSM794484_C1_R2_2.fq.gz
                      GSM794485_C1_R3_1.fq.gz
                      GSM794485_C1_R3_2.fq.gz
                      GSM794486_C2_R1_1.fq.gz
                      GSM794486_C2_R1_2.fq.gz
                      GSM794487_C2_R2_1.fq.gz
                      GSM794487_C2_R2_2.fq.gz
                      GSM794488_C2_R3_1.fq.gz
                      GSM794488_C2_R3_2.fq.gz
                      make_d_melanogaster_fb5_22.sh
                      README.txt
                      tophat_out

                      and I used this command:
                      tophat -p 8 -G genes.gtf -o C1_R1_thout--library-type=fr-firststrand\ genome C1_R1_1.fq C1_R1_2.fq

                      Thanks.

                      Comment


                      • #71
                        I have put together a tutorial website with four core tutorials on it, RNA-Seq, ChIP-Seq, Genome assembly, and SNP calling that may be of use to you.

                        This website was created to share bioinformatics tutorials and create a dynamic learning environment that does not become dated, PDF contributions welcome and there are four core tutorials available. We would be interested to get some feedback.

                        http://elvis.ccc.cranfield.ac.uk/CUB...gin-page.xhtml

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