Hello Everyone,
Recently, I am working with SOLiD RNA-seq short reads data. The reads are pair-ended with F3: 75bps and F5: 35bp. Now I am struggling with the question that which alignment tools should I choose.
Basically our goal is to perform alignment and expression summarization followed by differential expression analysis. In particular, we wish to be able to identify some novel transcripts or novel splicing events.
I have worked with Illumina short reads data on the same species using Tophat+Cufflinks before and they gave me reasonable results. Tophat successfully handled the splicing junctions and around 80% reads can be mapped to the genome.
With the SOLiD data on Tophat, I got only around 20% reads mapped. For example, when I aligned one of my libraries to the transcriptome (the first step in Tophat), only 4.64% reads were mapped. Then among these unmapped reads, only 19.92% can be mapped to the genome. The chopped segments had a mapping rate around 19% on junctions.
I assumed there must be something wrong with my parameters settings. I searched the forum and tuned a few parameters as suggested (basically allow more mis-matches). However, I got similar results with low mapping rate.
I am wondering if there are any alternative tools that can perform splice junction-aware alignment. I have read BFAST and SHRiMP from other posts, but it seems that none of them support the splice junctions or novel transcripts discovery.
Thanks a lot,
Zheng
Recently, I am working with SOLiD RNA-seq short reads data. The reads are pair-ended with F3: 75bps and F5: 35bp. Now I am struggling with the question that which alignment tools should I choose.
Basically our goal is to perform alignment and expression summarization followed by differential expression analysis. In particular, we wish to be able to identify some novel transcripts or novel splicing events.
I have worked with Illumina short reads data on the same species using Tophat+Cufflinks before and they gave me reasonable results. Tophat successfully handled the splicing junctions and around 80% reads can be mapped to the genome.
With the SOLiD data on Tophat, I got only around 20% reads mapped. For example, when I aligned one of my libraries to the transcriptome (the first step in Tophat), only 4.64% reads were mapped. Then among these unmapped reads, only 19.92% can be mapped to the genome. The chopped segments had a mapping rate around 19% on junctions.
I assumed there must be something wrong with my parameters settings. I searched the forum and tuned a few parameters as suggested (basically allow more mis-matches). However, I got similar results with low mapping rate.
I am wondering if there are any alternative tools that can perform splice junction-aware alignment. I have read BFAST and SHRiMP from other posts, but it seems that none of them support the splice junctions or novel transcripts discovery.
Thanks a lot,
Zheng
Comment