Simple Tophat question

kwatts59 · 06-02-2011, 06:03 PM

I am using Tophat to perform single-end alignment to a plant genome that has 12 chromosomes. Tophat generated a file called accepted_hits.bam (1.75G). I converted the file to a bed file using the bamToBed utility.
Then I split the bed file based on chromosomes. Below are the number of lines (reads) for each chromosome.
chrom1 - 4.9M
chrom2 - 6.2M
chrom3 - 5.0M
chrom4 - 2.4M
chrom5 - 5.3M
chrom6 - 2.8M
chrom7 - 3.0M
chrom8 - 2.6M
chrom9 - 12.6M
chrom10 - 1.7M
chrom11 - 1.8M
chrom12 - 1.8M

My question: Why are there 12.6 million reads on chromosome 9?
I looked at the genome map and chromosome 9 is the second smallest (chrom 10 is the smallest).

ishmael · 06-02-2011, 06:23 PM

I think you should check the data first.
Perhaps some high abundant sequences take large proportion of the dataset.(For example, rRNA reducing failed, so some rRNA occupied many reads?)
Check the density distribution of the reads at the chromosomes, it may provide some clues.

jmartin127 · 06-02-2011, 07:31 PM

Welcome to RNA-Seq. You may want to first forget everything you learned about genome sequencing.

I would align the reads to the transcriptome FASTA file, assuming you have one, and check which genes are most highly expressed, and if they are on chromosome 9. The size of the chromosome may be irrelevant. It could be that chromosome 9 is much more gene dense than the other chromosomes. Ishmael's suggestion is also good.

Quote:

Originally Posted by kwatts59

My question: Why are there 12.6 million reads on chromosome 9?
I looked at the genome map and chromosome 9 is the second smallest (chrom 10 is the smallest).

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06-02-2011, 06:03 PM	#1
kwatts59 Member Location: nevada Join Date: Apr 2011 Posts: 46	Simple Tophat question I am using Tophat to perform single-end alignment to a plant genome that has 12 chromosomes. Tophat generated a file called accepted_hits.bam (1.75G). I converted the file to a bed file using the bamToBed utility. Then I split the bed file based on chromosomes. Below are the number of lines (reads) for each chromosome. chrom1 - 4.9M chrom2 - 6.2M chrom3 - 5.0M chrom4 - 2.4M chrom5 - 5.3M chrom6 - 2.8M chrom7 - 3.0M chrom8 - 2.6M chrom9 - 12.6M chrom10 - 1.7M chrom11 - 1.8M chrom12 - 1.8M My question: Why are there 12.6 million reads on chromosome 9? I looked at the genome map and chromosome 9 is the second smallest (chrom 10 is the smallest).

06-02-2011, 06:23 PM	#2
ishmael Member Location: NY, US Join Date: Jul 2008 Posts: 17	I think you should check the data first. Perhaps some high abundant sequences take large proportion of the dataset.(For example, rRNA reducing failed, so some rRNA occupied many reads?) Check the density distribution of the reads at the chromosomes, it may provide some clues.