We have developed a computational method that counts the frequencies of unique k-mers in FASTQformatted genome data and uses this information to infer the genotypes of known variants. FastGT can detect the variants in a 30x genome in less than 1 hour using ordinary low-cost server hardware. The overall concordance with the genotypes of two Illumina "Platinum" genomes is 99.96%, and the concordance with the genotypes of the Illumina HumanOmniExpress is 99.82%. Our method provides k-mer database that can be used for the simultaneous genotyping of approximately 30 million single nucleotide variants (SNVs), including >23,000 SNVs from Y chromosome. The source code of FastGT software is available at GitHub (https://github.com/bioinfo-ut/GenomeTester4/). Next-generation sequencing (NGS) technologies are widely used for studying genome variation. Variants in the human genome are typically detected by mapping sequenced reads and then performing genotype calling [1] [2] [3] [4] . A standard pipeline requires 40-50 h to process a human genome with 30x coverage from raw sequence data to variant calls on a multi-thread server. Mapping and calling are state-of-the-art processes that require expert users familiar with numerous available software options. It is not surprising that different pipelines generate slightly different genotype calls [5] [6] [7] [8] [9] . Fortunately, inconsistent genotype calls are associated with certain genomic regions only [10] [11] [12] , whereas genotyping in the remaining 80-90% of the genome is robust and reliable. The use of k-mers (substrings of length k) in genome analyses has increased because computers can handle large volumes of sequencing data more efficiently. For example, phylogenetic trees of all known bacteria can be easily built using k-mers from their genomic DNA [13] [14] [15] . Bacterial strains can be quickly identified from metagenomic data by searching for strain-specific k-mers [16] [17] [18] . K-mers have also been used to correct sequencing errors in raw reads [19] [20] [21] [22] . One recent publication has described an alignment-free SNV calling method that is based on counting the frequency of k-mers 23 . This method converts sequences from raw reads into Burrows-Wheeler transform and then calls genotypes by counting using a variable-length unique substring surrounding the variant. We developed a new method that offers the possibility of directly genotyping known variants from NGS data by counting unique k-mers. The method only uses reliable regions of the genome and is approximately 1-2 orders of magnitude faster than traditional mapping-based genotype detection. Thus, it is ideally suited for a fast, preliminary analysis of a subset of markers before the full-scale analysis is finished. The method is implemented in the C programming language and is available as the FastGT software package. FastGT is currently limited to the calling of previously known genomic variants because specific k-mers must be pre-selected for all known alleles. Therefore, it is not a substitute for traditional mapping and variant calling but a complementary method that facilitates certain aspects of NGS-based genome analyses. In fact, FastGT is comparable to a large digital microarray that uses NGS data as an input. Our method is based on three original components: (1) the procedure for the selection of unique k-mers, (2) the customized data structure for storing and counting k-mers directly from a FASTQ file, and (3) a maximum likelihood method designed specifically for estimating genotypes from k-mer counts. Compilation of the database of unique k-mer pairs. The crucial component of FastGT is a pre-compiled flat-file database of genomic variants and corresponding k-mer pairs that overlap with each variant. Every bi-allelic single nucleotide variant (SNV) position in the genome is covered by k k-mer pairs, where pair is formed
FastGT REF is yet another k-mer-based method to genotype sequencing data: it strongly relies on a pre-compiled database of bi-allelic SNVs and corresponding k-mers, obtained by subjecting the k-mers that overlap known SNVs to several filtering steps.
FastGT: an alignment-free method for calling common SNVs directly from raw sequencing reads
Scientific Reports
Scientific Reports
Biology Medicine