2 answers
Hi, I am running a similar project. First to create reference transcriptome, you should assemble the reads by de novo assembler such asTrinity and Bridger. I guess you did this step because deferentially expressed genes are available to you. Then, the second step is to create supertranscripts and annotation file, if your assembler was Trinity, the following link (GATK pipeline in trinity pipeline) will be helpful for you: https://github.com/trinityrnaseq/trinityrnaseq/wiki/Variant-Calling Otherwise (other assemblers), you can use lace software to create supertranscripts and annotation file using reference transcriptome. Then, the GATK pipeline is used to create the vcf file.
Dear sam Sorry for the delayed response. Star is a splice aware aligner and is suitable for the snp calling step. I am not sure. Perhaps other splice aware aligner, such as tophat, may also be appropriate. CLC is a comprehensive analysis package and very useful. I didn’t work with it and I don’t know the details and which aligner it uses at this step.
Please elaborate further on what you are aiming to do, and be more specific on what data you currently have.
If you have X number of bulk RNA-seq samples, you could technically create a consensus transcriptome via de novo assembly and then find a way to perform variant calling in this way. Be wary of the pitfalls of using RNA-seq data for variant calling, though: A: Inferring genotype based on RNA sequnces
Kevin
I want to investigate the SNVs and indels of differentially expressed genes between various genotypes of the same species of plant (18 RNA-seq samples). The genotypes are divided in 2 groups according to their differences of specific metabolites content. Unfortunatley the genome is not available yet.
Cool - thanks. Then, perhaps, first creating a reference transcriptome via de novo assembly may be a good starting point.
I don't recommend variant calling on RNA-seq data, unless one is very familiar with the pitfalls of doing this ( see my link ).
GATK has a pipeline for it, but it's not well tested, as far as I know.
Please do look at the solution mentioned by eyonesi, too. I have not used Trinity but they may have a good solution for de novo assembly and variant calling.
Log in to answer this question.
-
Using Specific Regions of Genome as Reference for Alignment Using STAR
written by hkarakurt
-
how to use mirdeep2 without a small RNA library
written by Beatriz •
-
Integrate chip-seq with rna-seq?
written by Tonkatsu •
-
How to evaluate the performance of an RNA-seq pipeline
written by nhaus •
-
How to map a set of genes on a circular representation of DNA ?
written by CriticalPeriod •
-
Variant calling from RNA-seq?
written by zizigolu
-
annotation and genetic variants
written by deeksha3696 •
-
Tool to analysis GO term enrichment in C. elegans
written by biomagician
-
Detection of DE genes among different tissues of an organism during time - RNA seq
written by statfa
-
RNa seq data: GO annotation
written by hnp21 •
