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 hour 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 are 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 are 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 PacBiobased assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life.

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

“…The stLFR library construction requires ≥10 ng of high molecular weight DNA. PacBio SMRT sequencing has a high genomic DNA input requirement 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 [ 54 ]. Furthermore, PacBio recently released an amplification-based ultra-low DNA input protocol starting with 5 ng of high molecular weight DNA.…”

Radiation with reticulation marks the origin of a major malaria vector

“…New advances in sequencing technologies (e.g., ref. 68 ) will lead to improved assemblies that may allow further insights into the distribution of introgression blocks along the genome and the identification of adaptive introgression. Whether introgression has played a wider role in the origin of other dominant malaria vectors beyond An.…”

Novel genomic resources for shelled pteropods: a draft genome and target capture probes for Limacina bulimoides, tested for cross-species relevance

“…In order to overcome this challenge, genome sequencing projects should combine both short and long reads to resolve repetitive regions that span across short reads [54,55]. Single molecule real time (SMRT) sequencing techniques which produce long reads recommend substantial DNA input, although some recent developments in library preparation techniques have lowered the required amount of DNA [56]. These SMRT techniques also tend to be high in cost, which may be a limiting factor when choosing between sequencing methods.…”