Extract Transcript sequence from fastq file (Plant RNA-Seq data)

Question

[hafiz.talhamalik]

I have a plant RNA-Seq data and I want to extract the transcript sequence of the some transcripts. How can I do that ??

[cpad0112]

@ hafiz.talhamalik

Is this plant genome already sequenced and annotated? If so, go for reference based assembly (with reference sequence) and post alignment, you can extract transcripts (sequence) of interest using annotation file. For aligning RNAseq data, use splice aware aligners such as hisat2, star.

[hafiz.talhamalik]

yes it's known one Brassica Napus. I tried reference based assembly using trinity but assembly results were not good..!! any other good assembler ??

[cpad0112]

I guess Trinity is for de novo assembly. Since reference genome and annotation are available, use reference based assembly. Authors in this paper (on B. napus transcriptome) https://bmcgenomics.biomedcentral.com/articles/10.1186/s12864-015-2062-7 used tophat. I would suggest hisat2 or star. You can use reference genome and annotation from here: https://plants.ensembl.org/Brassica_napus/Info/Index @ hafiz.talhamalik

[ashish]

You can extract transcript sequences directly from ensembl plants biomart, you don't need RNA-seq data for that. In case it is transgenic with fused transcripts or something like that, then you can use blast to extract reads from fastq file and then assemble them.

[hafiz.talhamalik]

ok let me try that.. and any idea of tool for annotation of plant assembly ?

[ashish]

MAKER-P is good.

Answer 1

[Kristoffer Vitting-Seerup]

If you have RNAseq you can also do a guided transcriptome assembly - basically look for transcripts in your data that does not appear in the annotation database - you can read more about considerations and tools here.