Genotyping by next-generation sequencing

A method for genotyping-by-sequencing a sample, the method comprising:

• 1) ligating a first single-stranded oligonucleotide comprising a first primer binding site to the first ends of nucleic acid fragments produced by digesting nucleic acids from the sample with a restriction enzyme and ligating a second oligonucleotide comprising a second primer binding site to the second ends of the nucleic acid fragments to produce a plurality of templates;
• 2) amplifying templates using a first amplification primer that hybridizes to the first primer binding site or complement thereof and a second amplification primer that hybridizes to the second primer binding site or complement thereof to produce amplicons;
• 3) acquiring sequence reads from the amplicons;
• and
• 4) genotyping-by-sequencing the sample by determining a genotype of the sample using one or more sequence reads.

A method for genotyping-by-sequencing a plurality of samples, the method comprising:

• 1) ligating a first single-stranded oligonucleotide comprising a primer binding site and a first distinct barcode to the first ends of first nucleic acid fragments produced by digesting nucleic acids from a first sample with a restriction enzyme and ligating a common oligonucleotide to the second ends of the first nucleic acid fragments from the first sample to produce a first plurality of templates;
• 2) ligating a second single-stranded oligonucleotide comprising the primer binding site and a second distinct barcode to the first ends of second nucleic acid fragments produced by digesting nucleic acids from a second sample with the restriction enzyme and ligating a common oligonucleotide to the second ends of the second nucleic acid fragments from the second sample to produce a second plurality of templates;
• 3) amplifying templates from a template mixture comprising the first plurality of templates mixed with the second plurality of templates using a first amplification primer comprising a 5′ portion that hybridizes to the primer binding site or complement thereof and a second amplification primer that hybridizes to the common oligonucleotide or complement thereof to produce amplicons;
• 4) acquiring sequence reads from the amplicons;
• 5) associating sequence reads comprising a sequence of the first barcode with the first sample and associating sequence reads comprising a sequence of the second barcode with the second sample;
• and
• 6) genotyping-by-sequencing the first sample by determining a genotype of the first sample using one or more sequence reads and genotyping-by-sequencing the second sample by determining a genotype of the second sample using one or more sequence reads.