Hello all Seqanswer community Users,
I am biologist. Learning bioinformatics from scratch. Performing RNA-seq analysis for first time:
I got fastq files from ion proton instrument, it has single end read, 50-340 sequence length.
I don't have reference genome.
Here is how I did my analysis:
1]Fastqc
2]Trimmmimg some read using fastx tool:
a]First used fastq_qulaity trimmer -Q33 -t 20 -l 50 -i -o, because some of sequence has quality less than 18
b]fastx_trimmer = to trim off few reads from end of seq
For de novo assembly:
3]Velveth with kmer 31
4]velvetg
5] bowtie-build== to build reference index from contig.fa file created from velvetg
6] Mapping my fastq read with above build reference index
So my questions are:
1] Am i doing trimimg in right manner
2] On what basis you select parameter of velvetg and velveth
2] Which kmer value to select
3] Am I running bowtie in correct manner?
4] If yes, how do i confirm assembly created using velvet contain preserved input information and it's accuracy.
Hope you all can help me out.
Thanks a bunch in advance.
Naresh
I am biologist. Learning bioinformatics from scratch. Performing RNA-seq analysis for first time:
I got fastq files from ion proton instrument, it has single end read, 50-340 sequence length.
I don't have reference genome.
Here is how I did my analysis:
1]Fastqc
2]Trimmmimg some read using fastx tool:
a]First used fastq_qulaity trimmer -Q33 -t 20 -l 50 -i -o, because some of sequence has quality less than 18
b]fastx_trimmer = to trim off few reads from end of seq
For de novo assembly:
3]Velveth with kmer 31
4]velvetg
5] bowtie-build== to build reference index from contig.fa file created from velvetg
6] Mapping my fastq read with above build reference index
So my questions are:
1] Am i doing trimimg in right manner
2] On what basis you select parameter of velvetg and velveth
2] Which kmer value to select
3] Am I running bowtie in correct manner?
4] If yes, how do i confirm assembly created using velvet contain preserved input information and it's accuracy.
Hope you all can help me out.
Thanks a bunch in advance.
Naresh
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