Genotyping of polyploids
The current invention pertains to a reliable method for determining the relative frequency of a sequence variant of interest in a nucleic acid sample derived from at least one polyploid cell, wherein the method uses a UMI to correct for any amplification biases. The invention further pertains to the use of a UMI for accurately determining the relative frequency of a sequence variant of interest in a nucleic acid sample derived from at least one polyploid cell.
1 . A method for determining the genotype of a polyploid cell by determining the relative frequency of an allelic variant of interest in a nucleic acid sample, wherein the nucleic acid sample comprises genomic DNA derived from the polyploid cell, wherein the method comprises:
(a) providing polynucleotides derived from the nucleic acid sample, wherein the polynucleotides comprise the sequence of the allelic variant of interest or the reversed complement thereof, and wherein each polynucleotide comprises a unique molecular index (UMI),
(b) amplifying the polynucleotides provided in (a);
(c) determining the sequences of the amplified polynucleotides to obtain sequence reads;
(d) grouping the sequence reads using at least the UMI and obtaining a consensus sequence of the grouped sequence reads; and
(e) determining the genotype of the polyploid cell by calculating the number of times the allelic variant of interest occurs in the nucleic acid sample in relation to the total of the number of times all allelic variants thereof occur in the nucleic acid sample.
2 . The method according to claim 1 , wherein the polynucleotides of (a) are at least one of:
(i) fragments of nucleic acids from the sample, wherein each fragment is attached to a UMI; and
(ii) ligation products of probes capable of hybridizing to the allelic variant of interest in a nucleic acid from the sample, wherein each ligation product comprises a UMI.
3 . The method according to claim 1 , wherein (d) comprises collapsing sequence reads obtained in (c).
4 . The method according to claim 1 , wherein the allelic variant is present on a single locus.
5 . The method according to claim 1 , wherein the method is preceded by determining the ploidy level of the at least one polyploid cell.
6 . The method according to claim 1 , wherein the relative frequency of two or more allelic variants of interest is determined.
7 . The method according to claim 2 , wherein the polynucleotides are ligation products, and wherein the UMI is present in an allele-specific oligonucleotide ligation probe.
8 . The method according to claim 1 , wherein prior to sequencing (c), the polynucleotides or amplified polynucleotides are enriched.
9 . The method according to claim 8 , wherein polynucleotides or amplified polynucleotides are enriched using a hybridization-based capture method.
10 . The method according to claim 1 , wherein the polynucleotides further comprise a sample identifier.
11 . The method according to claim 2 , wherein the polynucleotides are fragments, and wherein at least a first adapter is ligated to the fragments and wherein the UMI is located in the first adapter, wherein optionally a second adapter is ligated to the fragment.
12 . The method according to claim 11 , wherein a sample identifier is present in the first or optional second adapter.
13 . The method according to claim 1 , wherein the method is multiplexed.