A High-Quality De novo Genome Assembly from a Single Mosquito Using PacBio Sequencing

Abstract

A high-quality reference genome is a fundamental resource for functional genetics, comparative genomics, and population genomics, and is increasingly important for conservation biology. PacBio Single Molecule, Real-Time (SMRT) sequencing generates long reads with uniform coverage and high consensus accuracy, making it a powerful technology for de novo genome assembly. Improvements in throughput and concomitant reductions in cost have made PacBio an attractive core technology for many large genome initiatives, however, relatively high DNA input requirements (~5 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that have lower DNA content, or on projects with limited input DNA for other reasons. Here we present a high-quality de novo genome assembly from a single Anopheles coluzzii mosquito. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 100 ng of starting genomic DNA. The sample was run on the Sequel System with chemistry 3.0 and software v6.0, generating, on average, 25 Gb of sequence per SMRT Cell with 20 h movies, followed by diploid de novo genome assembly with FALCON-Unzip. The resulting curated assembly had high contiguity (contig N50 3.5 Mb) and completeness (more than 98% of conserved genes were present and full-length). In addition, this single-insect assembly now places 667 (>90%) of formerly unplaced genes into their appropriate chromosomal contexts in the AgamP4 PEST reference. We were also able to resolve maternal and paternal haplotypes for over 1/3 of the genome. By sequencing and assembling material from a single diploid individual, only two haplotypes were present, simplifying the assembly process compared to samples from multiple pooled individuals. The method presented here can be applied to samples with starting DNA amounts as low as 100 ng per 1 Gb genome size. This new low-input approach puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

Extensive genomic and transcriptomic variation defines the chromosome-scale assembly ofHaemonchus contortus, a model gastrointestinal worm

“…The absence in the literature to date of such chromosome-scale assemblies for physically small and genetically diverse eukaryotic species likely reflects the significant investment required to improve a draft assembly to chromosomes. However, it is likely that as DNA input requirements for long-read sequencing decrease [56] , the ability and ease with which highly contiguous genomes can be generated for small and genetically diverse species will be significantly enhanced.…”

A High-Quality Genome Assembly from a Single, Field-collected Spotted Lanternfly (Lycorma delicatula) using the PacBio Sequel II System

“…L. delicatula is known to harbor several endosymbionts in specialized bacteriocytes, predominantly in the distal end of the insect abdomen; to avoid a high proportion of these symbionts in the sequencing, DNA was extracted from the head and thorax regions of the insect only (see Materials & Methods for details). While more recently developed single arthropod assemblies have significantly lowered DNA input requirements [24], here the amount of extracted genomic DNA was more plentiful because of the relatively larger size, allowing for sufficient DNA for a standard library preparation with size selection, resulting in a ~20 kb average insert size sequencing library ( Figure 2). The library was sequenced on the Sequel II System with one SMRT Cell 8M, yielding 131.6 Gb of total sequence contained in 5,639,857 reads, with a polymerase read length N50 of 41.7 kb and insert (subread) length N50 of 22.3 kb ( Figure S1).…”

“…Moreover, genomic regions with high heterozygosity tend to be assembled into more fragmented contigs [20], so computational methods specifically developed for heterozygous samples are needed [21][22][23]. Recently, high-quality long-read assemblies have been published for a single diploid mosquito (Anopheles coluzzii) [24] and a single haploid honeybee (Apis mellifera) [7]. Despite both species having relatively small genomes (<300 Mb), multiple PacBio SMRT Cells were needed for sufficient sequencing coverage (N=3 for mosquito, N=29 for bee).…”

Comparison of long read methods for sequencing and assembly of a plant genome

“…The stLFR library construction requires at least 10 ng of high molecular weight DNA. PacBio SMRT sequencing has a high genomic DNA input requirements of 5-20 µg of high molecular weight DNA for standard library protocol depending on the genome size but the PacBio low DNA input protocol has reduced this requirement to as low as 100 ng per 1 Gb genome size (45). Furthermore, PacBio recently released an amplification-based ultra-low DNA input protocol starting with 5 ng of high molecular weight DNA.…”