how to fetch the specific sequence of gene from rna seq data

Question

[fatimarasool135]

can we retrived the spesific seqence of the gene from rna seq data of one species.?

For this pupose i perform the following steps but the resulyt out put file contain long string of nnnn with nuclotide sequnce

Indexing by bowtie2

bowtie2-build --large-index -f  wheat.fa  wheat

mapping by tophat

tophat -p 30 -G wheat.gff3  wheat  G1_cleaned_R1.fastq  G1_cleaned_R2.fastq -o Alingment

Get consensus fastq file

samtools mpileup -uf REFERENCE.fasta  Aceepted_hits.bam | bcftools call -c | vcfutils.pl vcf2fq > batis_cns.fastq

Convert .fastq to .fasta

seqtk seq -aQ64 baris_cns.fastq > batis_cns.fasta

cordinate fetching

zcat Triticum_aestivum.gff3.gz |grep "TraesCS5A02G213300" > coordinate.bed12

Command for sequence fetching

bedtools getfasta -fi batis_cns.fasta -bed coordinate.bed12 -fo OUTPUT-GENE-SEQUENCE.fa

[finswimmer]

Hello fatimarasool135 ,

• Please use the formatting bar (especially the code option) to present your post better. I've done it for you this time.
  

• You should know that the old 'Tuxedo' pipeline of Tophat(2) and Cufflinks is no longer the "advisable" tool for RNA-seq analysis. The software is deprecated/ in low maintenance and should be replaced by HISAT2, StringTie and ballgown. See this paper: Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. There are also other alternatives, including alignment with STAR and bbmap, or pseudo-alignment using salmon.

Please stop using Tophat https://t.co/Es4ohxOEyx Cole and I developed the method in *2008*. It was greatly improved in TopHat2 then HISAT & HISAT2. There is no reason to use it anymore. I have been saying this for years yet it has more citations this year than last #methodsmatter

— Lior Pachter (@lpachter) December 2, 2017

Thank you!

[fatimarasool135]

Thank you please tell me steps to get the specific sequence of gene from rna seq data .like there is a gene abc in wheat i want to retrieve this abc from mine sample of rna seq .

[GenoMax]

You have described the procedure to get the consensus sequence for the gene you are interested in in your original post. So is there a question beyond that?

[fatimarasool135]

I simply follow these step to get gene sequence.... i want to know how i fetch the desired seq of gene from rna seq of mine sample

[fatimarasool135]

Hi. what will the reference sequence used for mapping of rna seq read ? is it shuold be cDNA or genomic sequence?

[GenoMax]

Both can potentially be used. If you are not interested in discovery of novel transcripts you could map against transcriptome ( and if you are using salmon or kallisto you would go this route). General recommendation is to align against the genome and then use a GTF (gene model file) to count reads that fall under the boundaries defined in that file.

[fatimarasool135]

from where i can got GTF file ?

[GenoMax]

Looks like you have a GFF3 file. That can work.

[fatimarasool135]

i have to generate it from tool or download it from ensembl ?