Long-read sequence assembly: a technical evaluation in barley

Abstract

Sequence assembly of large and repeat-rich plant genomes has been challenging, requiring substantial computational resources and often several complementary sequence assembly and genome mapping approaches. The recent development of fast and accurate long-read sequencing by circular consensus sequencing (CCS) on the PacBio platform may greatly increase the scope of plant pan-genome projects. Here, we compare current long-read sequencing platforms regarding their ability to rapidly generate contiguous sequence assemblies in pan-genome studies of barley (Hordeum vulgare). Most long-read assemblies are clearly superior to the current barley reference sequence based on short-reads. Assemblies derived from accurate long reads excel in most metrics, but the CCS approach was the most cost-effective strategy for assembling tens of barley genomes. A downsampling analysis indicated that 20-fold CCS coverage can yield very good sequence assemblies, while even 5-fold CCS data may capture the complete sequence of most genes. We present an updated reference genome assembly for barley with near-complete representation of the repeat-rich intergenic space. Long-read assembly can underpin the construction of accurate and complete sequences of multiple genomes of a species to build pan-genome infrastructures in Triticeae crops and their wild relatives.

Genome-Wide Analysis of NLR Disease Resistance Genes in an Updated Reference Genome of Barley

“…Since similar methodologies were used by our and previous studies ( Andersen et al, 2016 ; Habachi-Houimli et al, 2018 ), the result suggested that the great difference of NLR gene numbers in barley identified in the present study and previous studies should be caused by genome assembling issues of the short-read sequence strategy. This is in accordance with the result of a recent study, which showed that the updated barley genome has more NLR genes at the MLA locus than the early version genome assembly ( Mascher et al, 2021 ). Furthermore, the wide distribution of barley NLR genes on the phylogeny that constructed with NLR genes from T. urartu and A. thaliana suggested that NLR gene diversity in barley is also comparable with those in T. urartu .…”

“…The hexaploid wheat T. aestivum has over 2000 NLR genes due to recently occurred polyploidization, whereas the diploid wheat T. urartu has 537 NLR genes. However, by improving the barley genome with long-read sequence strategy, a recently study revealed that the NLR gene number in barley might have been underestimated ( Mascher et al, 2021 ).…”

“…Morex V3. were downloaded from the electronic data archive library (e!DAL) 2 ( Mascher et al, 2021 ). The Arabidopsis thaliana NLR genes were retrieved from our previous study ( Zhang et al, 2016 ).…”

“…These numbers are much smaller than those in diploid wheat genomes, which have more than 500 NLR genes ( Liu et al, 2021 ). A recent study reported a newly assembly of barley genome of the cultivar Morex by long-read sequencing technology ( Mascher et al, 2021 ). Investigation of the gene composition in the MLA locus revealed that three tandem CNL genes at this locus were missed in the earlier assemblies but present in the new assembly ( Mascher et al, 2021 ).…”

“…A recent study reported a newly assembly of barley genome of the cultivar Morex by long-read sequencing technology ( Mascher et al, 2021 ). Investigation of the gene composition in the MLA locus revealed that three tandem CNL genes at this locus were missed in the earlier assemblies but present in the new assembly ( Mascher et al, 2021 ). The quality of genome assembly and annotation is critical to genome-wide analysis to gene families, especially to R genes with resembled repeats and duplicates.…”