Question: Bowtie-Build, Which File To Use Contigs.Fa Or Transcript.Fa
3
gravatar for nbvasani
6.2 years ago by
nbvasani230
United States
nbvasani230 wrote:

Hi Community user,

I have single-end reads fastq file. I have generated denovo assembly using velvet/oases. Now i need to check, how good is my assembly. So I am using aligner, bowtie. But I am confused which fasta file to use for creating bowtie index i.e. shall I used contigs.fa from velveg or transcript.fa file from oases. Do you guys prefer --best parameter option while running bowtie?

I would really appreciate your feedback and reponse.

Thanks, Naresh

bioinformatics rna-seq bowtie • 3.3k views
ADD COMMENTlink modified 6.2 years ago by Istvan Albert ♦♦ 81k • written 6.2 years ago by nbvasani230
3
gravatar for Istvan Albert
6.2 years ago by
Istvan Albert ♦♦ 81k
University Park, USA
Istvan Albert ♦♦ 81k wrote:

Use both and compare the results.

Plus use bwa-mem instead of bowtie, that way your reads will align across junctions even for the contigs.fa file.

ADD COMMENTlink written 6.2 years ago by Istvan Albert ♦♦ 81k

Thanks Albert. I will try that.

ADD REPLYlink written 6.2 years ago by nbvasani230

Hi Albert, I tried bowtie with contigs.fa file, but I got only 6% alignment. Now I am trying to run with transcripts.fa. Any suggestion why alignment rate is so low. Fastq file used for alignment was trimmed to get better quality.

Thanks in advance. Naresh

ADD REPLYlink written 6.2 years ago by nbvasani230

that's why I said use bwa-mem

ADD REPLYlink written 6.2 years ago by Istvan Albert ♦♦ 81k

ohh ok. Thanks.

ADD REPLYlink written 6.2 years ago by nbvasani230
1

Hi Albert,

As per your advice I used bwa-mem for alignment it work out very well. It think I got 79% align. Can you please check my flagstat output. If I am interpreting result in correct manner.

If it is 79% align can you please help me how can I improve alignment above 90% by adding more parameter.

I used following cmd:

##bwa index contigs.fa
##bwa mem contigs.fa CombineIonXpressRNA_009_NareshPool_Chip1_2_WT1_fastx_trimmer_from_quality_trimmer_file.fastq > aln-se.sam
##samtools view -bS aln-se.sam > aligned_reads.bam
##samtools flagstat aligned_reads.bam
34569162 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 duplicates
27555361 + 0 mapped (79.71%:-nan%)
0 + 0 paired in sequencing
0 + 0 read1
0 + 0 read2
0 + 0 properly paired (-nan%:-nan%)
0 + 0 with itself and mate mapped
0 + 0 singletons (-nan%:-nan%)
0 + 0 with mate mapped to a different chr
0 + 0 with mate mapped to a different chr (mapQ>=5)

Appreciate your help.

Thanks,
Naresh

ADD REPLYlink modified 5 weeks ago by RamRS24k • written 6.2 years ago by nbvasani230

there is no reason to expect a 90% alignment rate, some or many of your reads may not have ended up in the assembly

ADD REPLYlink written 6.2 years ago by Istvan Albert ♦♦ 81k

Thanks Albert! That mean I understand correctly, 79% was aligned from flagstat output?

ADD REPLYlink written 6.2 years ago by nbvasani230
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