Multiplex sequencing of bacterial artificial chromosomes for assembling complex plant genomes

Plant Biotechnology Journal
Sebastian BeierMartin Mascher

Abstract

Hierarchical shotgun sequencing remains the method of choice for assembling high-quality reference sequences of complex plant genomes. The efficient exploitation of current high-throughput technologies and powerful computational facilities for large-insert clone sequencing necessitates the sequencing and assembly of a large number of clones in parallel. We developed a multiplexed pipeline for shotgun sequencing and assembling individual bacterial artificial chromosomes (BACs) using the Illumina sequencing platform. We illustrate our approach by sequencing 668 barley BACs (Hordeum vulgare L.) in a single Illumina HiSeq 2000 lane. Using a newly designed parallelized computational pipeline, we obtained sequence assemblies of individual BACs that consist, on average, of eight sequence scaffolds and represent >98% of the genomic inserts. Our BAC assemblies are clearly superior to a whole-genome shotgun assembly regarding contiguity, completeness and the representation of the gene space. Our methods may be employed to rapidly obtain high-quality assemblies of a large number of clones to assemble map-based reference sequences of plant and animal species with complex genomes by sequencing along a minimum tiling path.

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Citations

Apr 28, 2017·Scientific Data·Sebastian BeierMartin Mascher
Nov 27, 2016·The Plant Journal : for Cell and Molecular Biology·Eva BauerUwe Scholz
Nov 18, 2018·TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik·Cécile MonatMartin Mascher
Sep 23, 2018·Genome Biology·Mona SchreiberMartin Mascher
Apr 16, 2021·G3 : Genes - Genomes - Genetics·Wayne XuAna Badea

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Datasets Mentioned

BETA
AF252830
AF285443
AY268139
371193

Methods Mentioned

BETA
Rice Genome Sequencing
Illumina sequencing
genotyping
PCR
Genome Sequencing
454 sequencing
Human
electrophoresis

Software Mentioned

sam2tab
samtools view ‐ q
SSPACE
clc
BEDtools
ThreadPoolExecuter
nucmer
BLASTN
MEM
R

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