PacBio Single-molecule Sequencing Research Articles

De novo genome assembly of the red silk cotton tree (Bombax ceiba).

Background Bombax ceiba L. (the red silk cotton tree) is a large deciduous tree that is distributed in tropical and sub-tropical Asia as well as northern Australia. It has great economic and ecological importance, with several applications in industry and traditional medicine in many Asian countries. To facilitate further utilization of this plant resource, we present here the draft genome sequence for B. ceiba.FindingsWe assembled a relatively intact genome of B. ceiba by using PacBio single-molecule sequencing and BioNano optical mapping technologies. The final draft genome is approximately 895 Mb long, with contig and scaffold N50 sizes of 1.0 Mb and 2.06 Mb, respectively.ConclusionsThe high-quality draft genome assembly of B. ceiba will be a valuable resource enabling further genetic improvement and more effective use of this tree species.

OR20 De novo generation of MHC haplotypes using single molecule sequencing

Aim Utilizing a previously developed long-fragment DNA capture methodology, PacBio single molecule real-time (SMRT) sequencing and a custom de novo assembly analysis pipeline, we aim to resolve the long-range, fully phased MHC haplotypes for a single heterozygous individual. Methods Long fragment DNA enrichment for the MHC was performed on fresh blood obtained from a heterozygous individual using our previously described method, RSE. Captured DNA was sequenced on the PacBio RSII sequencing platform. Raw reads were selected to include only those reads that map to annotated MHC haplotype sequences (n = 8). De novo assembly was performed using the CANU pipeline with tuned algorithm parameters to optimize continuity and accuracy of haplotigs (phased contigs) as compared to phased SNP genotyping data. Results Following de novo assembly, PacBio reads were assembled into 27 contiguous fragments (contigs) covering 97% of the 4Mbp MHC (as aligned to the COX MHC haplotype). Phased contigs (haplotigs) were found to agree with phased SNP genotyping data at ∼78% of interrogated positions (n = 4,779 probes) indicating that the majority of haplotigs are properly phased. Phased SNP genotyping data was also used to phase across breaks in the assembly, resulting in the generation of two distinct MHC haplotypes for the sequenced sample. Conclusions Our results demonstrate the capacity to generate de novo assembled, fully phased MHC haplotypes from PacBio single molecule sequencing reads. Although phasing across contig breaks caused by coverage gaps remains a challenge, we hope to resolve this issue using BioNano genomics optical maps, so as not to rely on phased SNP genotyping data. In essence, this method has the capacity to resolve full-length MHC haplotype sequences without the need for any reference genome or familial trio analysis.

Characterization of the Rosellinia necatrix Transcriptome and Genes Related to Pathogenesis by Single-Molecule mRNA Sequencing.

White root rot disease, caused by the pathogen Rosellinia necatrix, is one of the world’s most devastating plant fungal diseases and affects several commercially important species of fruit trees and crops. Recent global outbreaks of R. necatrix and advances in molecular techniques have both increased interest in this pathogen. However, the lack of information regarding the genomic structure and transcriptome of R. necatrix has been a barrier to the progress of functional genomic research and the control of this harmful pathogen. Here, we identified 10,616 novel full-length transcripts from the filamentous hyphal tissue of R. necatrix (KACC 40445 strain) using PacBio single-molecule sequencing technology. After annotation of the unigene sets, we selected 14 cell cycle-related genes, which are likely either positively or negatively involved in hyphal growth by cell cycle control. The expression of the selected genes was further compared between two strains that displayed different growth rates on nutritional media. Furthermore, we predicted pathogen-related effector genes and cell wall-degrading enzymes from the annotated gene sets. These results provide the most comprehensive transcriptomal resources for R. necatrix, and could facilitate functional genomics and further analyses of this important phytopathogen.

Improved Complete Genome Sequence of the Extremely Radioresistant Bacterium Deinococcus radiodurans R1 Obtained Using PacBio Single-Molecule Sequencing.

The genome sequence of Deinococcus radiodurans R1 was published in 1999. We resequenced D. radiodurans R1 using PacBio and compared the sequence with the published one. Large insertions and single nucleotide polymorphisms (SNPs) were observed among the genome sequences. A more accurate genome sequence will be helpful to studies of D. radiodurans.