AFLAP: Assembly-Free Linkage Analysis Pipeline usingk-mers from whole genome sequencing data

Abstract

BackgroundGenetic maps are an important resource for validation of genome assemblies, trait discovery, and breeding. Next generation sequencing has led to the production of high-density genetic maps constructed with 10,000s of markers. Most current approaches require a genome assembly of the organism under study, or a close relative, to identify markers. Our Assembly Free Linkage Analysis Pipeline (AFLAP) removes this requirement by using uniquely segregating k-mers as markers. This removes potential biases including preferential read alignment and variant calling.ResultsWe tested AFLAP using 100 F2 individuals of Arabidopsis thaliana, sequenced to low coverage. Genetic maps generated using k-mers specific for each parent contained over 130,000 markers that were concordant with the genomic assembly of the five chromosomes. AFLAP was then applied to 83 F1 individuals, sequenced to >5x coverage, generated from two crosses of the oomycete Bremia lactucae. The genetic map contained over 90,000 markers ordered in 19 large linkage groups. The genetic map was used to fragment, order, orient, and scaffold the reference assembly of B. lactucae, resulting in a much improved, genetically consistent genome assembly.ConclusionsAFLAP can therefore be used to generate high density linkage maps and improve draft genome assemblies of non-model organisms when a mapping population sequenced to adequate coverage is available. The genetic maps produced for B. lactucae were accurately aligned to the reference assembly and resulted in significant improvements of the assembly.

Ancestral chromosomes for the Peronosporaceae inferred from a telomere-to-telomere genome assembly of Peronospora effusa

“…Comparative genomics revealed a high level of synteny between P. effusa and the genetically orientated genome assembly of B. lactucae (Fletcher et al, 2020). The P. effusa chromosomes were numbered to match the corresponding chromosome-scale scaffolds of B. lactucae, reported previously (Fletcher et al, 2020), because they have highly conserved single-copy gene contents, similar gene orders along their chromosomes, and likely the same number of chromosomes (Fig.…”

“…4A, 4B). The genome of B. lactucae has been ordered into 19 linkage groups (Fletcher et al, 2020), 15 of which were colinear with complete chromosomes of P. effusa (Fig. 4A).…”

“…Preliminary analysis was conducted by transferring annotations from P. effusa R13 (Fletcher et al, 2018) onto the new assembly using Liftoff v1.3.0 (Shumate and Salzberg, 2020). Synteny of intermediate assemblies with the genetically oriented assembly of B. lactucae (GCA_004359215.2) (Fletcher et al, 2020) was determined using orthologs as calculated by OrthoFinder v2.2.1 (Emms and Kelly, 2015). The initial 12 chromosomes were found to be colinear with B. lactucae linkage group-spanning scaffolds.…”

“…Previous P. effusa genome assemblies (Feng et al, 2018a;Fletcher et al, 2020;Klein et al, 2020) were aligned to the new genome assembly using BLASTn (Altschul et al, 1990), filtering for alignments with a minimum length of 2,500 bp and scoring ≥95% identity. Genes for which 95% of the feature was within assembly-to-assembly alignments were reported as covered, regardless of the alignment length.…”

High-resolution genetic dissection of the major QTL for tipburn resistance in lettuce,Lactuca sativa

“…k-mers with a frequency lower than six were discarded to reduce noise introduced by sequencing error. k-mers unique to each pool were then extracted and assembled into contigs using the software ABySS ( Jackman et al 2017 ) with the parameters k = 25, c = 0, and e = 0 ( Fletcher et al 2020 ). Contigs longer than 1 Kb were mapped against the lettuce reference assembly.…”