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  • Help with Tablet

    So I have reads I have aligned to the mouse mm10 genome using Bowtie 2. I created a .bam from the .sam file, which I then used to make a sorted.bam and an indexed.bam.bai. I loaded the sorted.bam and indexed.bam.bai into tablet, and it's showing there are reads but nothing shows up on my viewer.

  • #2
    Are you loading a reference fasta along with the SAM/bam file? Tablet has options to load both.

    Didn't realize you could load the bai instead of the original fasta.

    Edit: Converted a SAM+FASTA I had on hand to sorted.bam/bai and sorted.bam/fasta...appears to work fine.

    Newest version of tablet? Can you run samtools flagstat on the bam file?

    Edit 2: Did you directly sequence mouse? If mouse is supposed to be low abundance, BAM files also load as chunks, so it's possible you may be visualizing a chunk of reference that is simply low abundance.
    Last edited by winsettz; 10-18-2013, 08:50 AM.

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    • #3
      Originally posted by winsettz View Post
      Are you loading a reference fasta along with the SAM/bam file? Tablet has options to load both.

      Didn't realize you could load the bai instead of the original fasta.

      Edit: Converted a SAM+FASTA I had on hand to sorted.bam/bai and sorted.bam/fasta...appears to work fine.

      Newest version of tablet? Can you run samtools flagstat on the bam file?

      Edit 2: Did you directly sequence mouse? If mouse is supposed to be low abundance, BAM files also load as chunks, so it's possible you may be visualizing a chunk of reference that is simply low abundance.
      So I was able to find reads after all; however, there was no annotation other than chromosome location. So I gave up on Tablet. Basically, I'm just trying to figure out my reads from RNAseq, so I've moved onto cufflinks. Hopefully, this works.

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      • #4
        Chromosome location and orientation is what you get from SAM/BAM.

        If you have any questions; don't forget seqanswers has an RNAseq forum!
        Last edited by winsettz; 10-18-2013, 10:19 AM.

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        • #5
          Basically I performed RNA sequencing with ~30mers using MiSeq. Now I just want to determine what genes are being transcribed. So aligned my reads to the mouse mm10 genome using bowtie 2. This outputs a Sam file so I converted to a sorted bam using samtools. All I want to do is see a list of transcripts so now I'm tryin cufflinks

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          • #6
            Originally posted by KnowNothing2 View Post
            So I was able to find reads after all; however, there was no annotation other than chromosome location. So I gave up on Tablet. Basically, I'm just trying to figure out my reads from RNAseq, so I've moved onto cufflinks. Hopefully, this works.
            As I had told you yesterday in a different thread the annotation will come from the gff/gtf file that I had linked. You need to have your bam file open along with the gff file in IGB/tablet/IGV in order to see the gene annotations.

            Working with NGS data is not simple (as you have discovered by now). You can't give up and keep hopping on to the next program. Nothing may work if you keep doing that. Stick with one program and then we can help you out.

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            • #7
              Originally posted by GenoMax View Post
              As I had told you yesterday in a different thread the annotation will come from the gff/gtf file that I had linked. You need to have your bam file open along with the gff file in IGB/tablet/IGV in order to see the gene annotations.

              Working with NGS data is not simple (as you have discovered by now). You can't give up and keep hopping on to the next program. Nothing may work if you keep doing that. Stick with one program and then we can help you out.
              Not easy, indeed.

              The reason I switched to a new program is because I indexed my bam file this morning and used that as the consensus file in Tablet. Maybe I'm not using Tablet correctly, but I scan down the chromosome and see my reads but no annotations. I even tried this using the gtf file you linked (see attached image)
              Attached Files

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              • #8
                Originally posted by KnowNothing2 View Post
                Not easy, indeed.

                The reason I switched to a new program is because I indexed my bam file this morning and used that as the consensus file in Tablet. Maybe I'm not using Tablet correctly, but I scan down the chromosome and see my reads but no annotations. I even tried this using the gtf file you linked (see attached image)
                It appears that Tablet can only take GFF3 format annotation files. So here is a link for mm10 annotation in GFF3 format: http://genes.mit.edu/burgelab/miso/a...0/ensGene.gff3 Download and save locally.

                Once you load your SAM/BAM file (along with your reference) in tablet, use the "import features" button (next to "open assembly") to point to this gff3 file. You should be able then jump to the features once they are imported in.

                Keep in mind that you are only "viewing" the alignments. Expression analysis (differential?) will come next.

                Comment


                • #9
                  Originally posted by GenoMax View Post
                  It appears that Tablet can only take GFF3 format annotation files. So here is a link for mm10 annotation in GFF3 format: http://genes.mit.edu/burgelab/miso/a...0/ensGene.gff3 Download and save locally.

                  Once you load your SAM/BAM file (along with your reference) in tablet, use the "import features" button (next to "open assembly") to point to this gff3 file. You should be able then jump to the features once they are imported in.

                  Keep in mind that you are only "viewing" the alignments. Expression analysis (differential?) will come next.
                  Awesome, thanks. So I now see if my reads represent an exon, which is very useful. Now to find out which gene that is, do I need to just manual search them?

                  Also, how did you find the GFF3 file? I googled it and but had no such luck finding it.
                  Attached Files

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