Separating homeologs by phasing in the tetraploid wheat transcriptome

Abstract

BackgroundThe high level of identity among duplicated homoeologous genomes in tetraploid pasta wheat presents substantial challenges for de novo transcriptome assembly. To solve this problem, we develop a specialized bioinformatics workflow that optimizes transcriptome assembly and separation of merged homoeologs. To evaluate our strategy, we sequence and assemble the transcriptome of one of the diploid ancestors of pasta wheat, and compare both assemblies with a benchmark set of 13,472 full-length, non-redundant bread wheat cDNAs.ResultsA total of 489 million 100 bp paired-end reads from tetraploid wheat assemble in 140,118 contigs, including 96% of the benchmark cDNAs. We used a comparative genomics approach to annotate 66,633 open reading frames. The multiple k-mer assembly strategy increases the proportion of cDNAs assembled full-length in a single contig by 22% relative to the best single k-mer size. Homoeologs are separated using a post-assembly pipeline that includes polymorphism identification, phasing of SNPs, read sorting, and re-assembly of phased reads. Using a reference set of genes, we determine that 98.7% of SNPs analyzed are correctly separated by phasing.ConclusionsOur study shows that de novo transcriptome assembly of tetraploid wheat benefit from multiple k-mer assembly strategies more than diploid wheat. Our results also demonstrate that phasing approaches originally designed for heterozygous diploid organisms can be used to separate the close homoeologous genomes of tetraploid wheat. The predicted tetraploid wheat proteome and gene models provide a valuable tool for the wheat research community and for those interested in comparative genomic studies.

expVIP: a Customizable RNA-seq Data Analysis and Visualization Platform

“…Analysis of a manually curated set of 52 tetraploid wheat homeologues showed that they share 97.3% 6 1.2% DNA sequence identity and that the distance between adjacent variants decreases exponentially, with an average separation of approximately 38 bp. This determines that 8% of singlenucleotide polymorphisms (SNPs) between A and B genome homeologous are over 100 bp apart (Krasileva et al, 2013). This would prevent reads containing these widely spaced SNPs from being unambiguously mapped to one homeologue, explaining why we observe a residual level of expression from the deleted chromosome in the nullitetrasomic lines.…”

“…To assess whether kallisto could correctly assign reads to the relevant homeologue, we used a unique genetic resource available in wheat: nullitetrasomic lines (Sears, 1954). Normal bread wheat contains three copies of most genes, one on each of the A, B, and D homeologous chromosomes, and these genes share over 95% identity in coding sequences (Krasileva et al, 2013). In nullitetrasomic lines, one chromosome is specifically deleted (nulli) and compensated by an additional copy of a homeologous chromosome (tetra).…”

The design of experiments for the transcriptome studies by high-throughput sequencing methods

“…Например, протяженность транскриптома дрозофилы ненамного меньше транскриптома у млекопитающих (Nfonsam et al, 2012). В то же время у злаковых средняя протяжен-ность транскриптома в два -три раза больше, чем у мыши (Krasileva et al, 2013), поэтому для этих видов растений целесообразно установить глубину секвенирования не менее чем в 4-5 × 10 9 п. н.…”

Next Generation Sequencing Enabled Genetics in Hexaploid Wheat

“…In the bulks, the individual reads align across the myriad of sources (Pontius et al 2002 ). More recently, we have shifted to a phased transcriptome which has gene models separated by the corresponding genome (Krasileva et al 2013 ).…”

“…We sequence the parental genotypes (Avocet S and Avocet S + Yr15 , Fig. 22.1a, b ) and generate a consensus reference by aligning the reads to the UniGenes and gene models described above (Krasileva et al 2013 ). Although genome-specifi c references are used when possible, there are still cases where multiple homoeologues will align to a common reference (as illustrated in Fig.…”