Single-Molecule Real-Time Sequencing Combined with Optical Mapping Yields Completely Finished Fungal Genome.
Next-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio-generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism's biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes.


OBJECTIVE
Studying whole-genome sequences has become an important aspect of biological research. The advent of next-generation sequencing (NGS) technologies has nowadays brought genomic science within reach of most research laboratories, including those that study nonmodel organisms. However, most genome sequencing initiatives typically yield (highly) fragmented genome assemblies. Nevertheless, considerable relevant information related to genome structure and evolution is likely hidden in those nonassembled regions. Here, we investigated a diverse set of strategies to obtain gapless genome assemblies, using the genome of a typical ascomycete fungus as the template. Eventually, we were able to show that a combination of PacBio-generated long reads and optical mapping yields a gapless telomere-to-telomere genome assembly, allowing in-depth genome analyses to facilitate functional studies into an organism's biology.
Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands.
Luigi
L
Faino
Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands.
Michael F
MF
Seidl
KeyGene N.V., Wageningen, The Netherlands.
Erwin
E
Datema
Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands.
Grardy C M
GC
van den Berg
KeyGene N.V., Wageningen, The Netherlands.
Antoine
A
Janssen
KeyGene N.V., Wageningen, The Netherlands.
Alexander H J
AH
Wittenberg
Laboratory of Phytopathology, Wageningen University, Wageningen, The Netherlands bart.thomma@wur.nl.
Bart P H J
http://orcid.org/0000-0003-4125-4181
BP
Thomma
D002874:Chromosome Mapping / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics; D016681:Genome, Fungal; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D056972:Optical Restriction Mapping / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods; D016615:Telomere / Q000235:genetics; D020172:Verticillium / Q000235:genetics*
D004251:DNA Transposable Elements