I have been struggling with de novo assembly of bacterial RNA-Seq data, but it is difficult for me to figure out how to use softwares and analyze the data because I have little background on bioinformatics or computer science. Some have suggested me to use free softwares such as Velvet and Trinity. But due to the issue of hardware and the convenience, I eventually used another software, called “Rockhopper” (http://cs.wellesley.edu/~btjaden/Rockhopper/), other than those popular softwares. "Rockhopper" uses de Bruijn graphs method.
Anyway, using “Rockhopper”, I obtained a total of 2,993 transcript sequences the sizes of which range from 50bp to 10,615bp. I have some questions at this point:
1. How can I align my sequencing data to this transcript sequence set to obtain read numbers? Can I just use typical mapping softwares such as “bowtie2”? I wondered this because the de novo assembled transcript set looks very different from a certain reference genome data (i.e., the typical bacterial reference genome has one long chromosomal sequence, whereas the de novo assembled transcript set has multiple, relatively short sequences). Once I get read numbers, I will do differential expression analysis by using DESeq2 package on R software.
2. How can I annotate each transcript? Is there any useful tutorial for this?
I hope to get a lot of advices from you guys.
Thanks.
Anyway, using “Rockhopper”, I obtained a total of 2,993 transcript sequences the sizes of which range from 50bp to 10,615bp. I have some questions at this point:
1. How can I align my sequencing data to this transcript sequence set to obtain read numbers? Can I just use typical mapping softwares such as “bowtie2”? I wondered this because the de novo assembled transcript set looks very different from a certain reference genome data (i.e., the typical bacterial reference genome has one long chromosomal sequence, whereas the de novo assembled transcript set has multiple, relatively short sequences). Once I get read numbers, I will do differential expression analysis by using DESeq2 package on R software.
2. How can I annotate each transcript? Is there any useful tutorial for this?
I hope to get a lot of advices from you guys.
Thanks.
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