cDNA database based on genome resequencing

Question

[boczniak767]

Hi,

I have some expression data acquired with old microarrays. The microarrays has been designed using ESTs from several maize lines, non-traceable.

Now, I have data from transcriptome profiling using these microarrays but for other maize line. The reference genome is B73 and I think most of probes has been done for it, though I can't prove it.

The natural thing would be mapping oligo probes to the B73 genome to see how the probes relate to new data on its sequence.

The problem is that I have done experiments with material from other line. So maybe it would be better to map oligos to transcripts of "my" line.

I have the genome of "my" line resequenced and corresponding vcf file. So I wonder if I can somehow use 1. the reference sequence (B73), 2. gff ot gtf file for it. 3. vcf with data for "my" line. to get cDNA sequences for "my" maize line?

I know, I can apply SNPs to genome of "my" line as follows bcftools consensus -i -s 140903_SND104_A_L007_HDS-1 -H 1 -f ../../agpv3/genome.fa AGPv3.fasta.mp.bcf.MUT.filt.vcf.gz -o s68_ref.fa cat ../../agpv3/genome.fa | vcf-consensus AGPv3.fasta.mp.bcf.MUT.filt_S68.vcf.gz > s68_ref.fa

Answer 1

[JC]

I think you already have your answer, my only comment is to be sure your VCF is only SNV data, otherwise, you will change coordinates.

Also, I believe you can directly map your reads to B73 transcripts and check coverages, if the variation is in a single SNP per transcript, it should not affect too much the mapping.

[boczniak767]

I only know, that with bcftools I can make pseudo-genome of my line based on vcf and reference line. Still I don't know how to make something similar with transcripts, i.e. to have new version of spliced cDNA.

[boczniak767]

if the variation is in a single SNP per transcript, it should not affect too much the mapping

The problem is that for some probes I have perfect match to transcript of one gene, but almost perfect match (one mismatch) for transcript of other gene. So I try to decide, how many mismatches are acceptable, and SNPs are complicating it.

[boczniak767]

@JC For a while I thought that I understand your answer. I.e. that I can apply SNPs to B73 genome and use gtf/gff to extract sequence with bedtools getfasta. But it is not possible, gtf contains coordinates of "transcripts" but to get cDNA I should rather join proper exons to avoid using also introns. I'm continuing my search for appropriate tool, so dear all feel free to comment/answer :-)

[JC]

is the GTF/GFF coordinates for your line or for B73?

[boczniak767]

My gff is for B73.

[boczniak767]

@JC I came across HGVS format and VEP tool which can write such format from vcf. It looks promising for me, but before I would get far into manual, could anybody tell me if above tools allow retrieval of transcripts with applied variants? And, if HGVS supports custom IDs, i.e. not ensembl or refseq