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June 1, 2021  |  

De novo PacBio long-read assembled avian genomes correct and add to genes important in neuroscience and conservation research

To test the impact of high-quality genome assemblies on biological research, we applied PacBio long-read sequencing in conjunction with the new, diploid-aware FALCON-Unzip assembler to a number of bird species. These included: the zebra finch, for which a consortium-generated, Sanger-based reference exists, to determine how the FALCON-Unzip assembly would compare to the current best references available; Anna’s hummingbird genome, which had been assembled with short-read sequencing methods as part of the Avian Phylogenomics phase I initiative; and two critically endangered bird species (kakapo and ‘alala) of high importance for conservations efforts, whose genomes had not previously been sequenced and assembled.


June 1, 2021  |  

A low DNA input protocol for high-quality PacBio de novo genome assemblies from single invertebrate individuals

A high-quality reference genome is an essential tool for studies of plant and animal genomics. 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. PacBio is the 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, non-inbred organisms that may have lower DNA content. Here we present high-quality de novo genome assemblies from single invertebrate individuals for two different species: the Anopheles coluzzii mosquito and the Schistosoma mansoni parasitic flatworm. A modified SMRTbell library construction protocol without DNA shearing and size selection was used to generate a SMRTbell library from just 50-100 ng of starting genomic DNA. The libraries were run on the Sequel System with chemistry v3.0 and software v6.0, generating a range of 21-32 Gb of sequence per SMRT Cell with 20 hour movies, and followed by diploid de novo genome assembly with FALCON-Unzip. The resulting assemblies had high contiguity (contig N50s over 3 Mb for both species) and completeness (as determined by conserved BUSCO gene analysis). We were also able to resolve maternal and paternal haplotypes for 1/3 of the genome in both cases. 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. This new low-input approach puts PacBio-based assemblies in reach for small, highly heterozygous organisms that comprise much of the diversity of life. The method presented here can be applied to samples with starting DNA amounts around 100 ng per 250 Mb – 1 Gb genome size.


June 1, 2021  |  

A low DNA input protocol for high-quality PacBio de novo genome assemblies

A high-quality reference genome is an essential tool for studying the genetics of traits and disease, organismal, comparative and conservation biology, and population genomics. 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 (3 µg for standard library protocol) have placed PacBio out of reach for many projects on small organisms that may have lower DNA content or on projects with limited input DNA for other reasons. Here we present a modified SMRTbell library construction protocol without DNA shearing or size selection that can be used to generate a SMRTbell library from just 150 ng of starting genomic DNA. Remarkably, the protocol enables high quality de novo assemblies from single invertebrate individuals and is applied to taxonomically diverse samples. 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 libraries were run on the Sequel System with chemistry v3.0 and software v6.0, generating ~11 Gb of sequence per SMRT Cell with 10 hour movies, and followed by de novo genome assembly with FALCON. The resulting assemblies had high contiguity (contig N50s over 1 Mb) and completeness (as determined by conserved BUSCO gene analysis) when at least 30-fold unique molecular coverage is obtained. This new low-input approach now puts PacBio-based assemblies in reach for small highly heterozygous organisms that comprise much of the diversity of life. The method presented here is scalable and can be applied to samples with starting DNA amounts of 150 ng per 300 Mb genome size.


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