RARSeq
RARSeq
RARseq is a cDNA-based, genotype-by-sequencing method for identifying RNA SNPs and polymorphic loci for population genomics. This method performs read alignment using sequencing data from single and dual restriction enzyme digestion libraries and aligns them using both de novo and reference-based genome assembly.
In RARseq, total RNA is isolated from samples. Next, first- and second-strand cDNA are generated by RT. The double-stranded cDNA is digested with selected restriction enzymes (MseI and MseI-StyI-HF). The double-stranded cDNA fragments are ligated to sequencing adapters before purification and size-selection to 200 bp. The samples are PCR-amplified, purified, and sequenced. Reads from both single- and double-digestion libraries are used for de novo and reference-based genome assembly.
Pros:
- Identifies RNA SNPs and polymorphic loci for population genomics studies
- Reduces reads from nongenic regions arising from highly methylated genomes
- Uses both de novo and reference-based genome assembly for read alignment
- More accurate SNP and allele discovery than genotyping by sequencing methods
- Restriction enzyme selection is flexible and can be customized, depending on the genome
Cons:
- Not yet adopted widely by the scientific community
- Has only been performed on plant genomes