Is it possible to get a gene sequence by genome re-sequecing?

Question

[Yao]

Hi everyone, I want to get a grape gene sequence by genome re-sequencing, but the material I have now is a backcross population ( [Vitis vinifera×Vitis amurensis] × Vitis vinifera ), which means they may highly heterozygous. I wonder if there would be chimerism genome assembly issue or any other problems when I try to pick some individuals and re-sequence them, and do there have any technique could help me? Thank you for your time and innovation!

Answer 1

[colindaven]

• Yes, highly heterozygous samples are a problem
• You can go for trio-binning using kmers from Illumina data from the parents, subdivide the long reads into two sets, then perform long read genome assembly (this is advanced, and you need Illumina AND long read data)
• Otherwise just get the longest reads you can get from either oxford nanopore tech Q20+ or Pacbio HiFi and assemble. You may require Illumina data for polishing.
• a suitable assembler might be flye or shasta

[Yao]

Thank you for your reply! I wanna know that if it was possible to get a gene 's real sequence by WGS, or PCR-based gene clone is the only way? ( I wanna know because some of the interested genes are really big and long (~5kb))

[GenoMax]

I wanna know that if it was possible to get a gene 's real sequence by WGS

Answer to that is a no, if you only want to sequence transcribed parts. If you are interested in plain sequence of the entire gene region then you can use WGS. But if you wish to find out sequence of the parts that are being transcribed then you would want to use isoSeq from PacBio or Nanopore sequencing to directly sequence full-length transcripts.

[colindaven]

Yes, 5kbp transcripts should be doable. PCR based amplicon methods are going to be a LOT cheaper though (maybe 2000-3000 € these days for a WGS on a promethion flowcell. But I think Vitis has a small genome so you might be able to get someone around you with a minion flowcell or two spare to do some sequencing.

You should get quite long contigs == long chunks of DNA from modern seq tech.

[Yao]

Thank you for your guys kindly reply, I have learned a lot :)