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September 21, 2019  |  

Assembling large genomes with single-molecule sequencing and locality-sensitive hashing.

Long-read, single-molecule real-time (SMRT) sequencing is routinely used to finish microbial genomes, but available assembly methods have not scaled well to larger genomes. We introduce the MinHash Alignment Process (MHAP) for overlapping noisy, long reads using probabilistic, locality-sensitive hashing. Integrating MHAP with the Celera Assembler enabled reference-grade de novo assemblies of Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and a human hydatidiform mole cell line (CHM1) from SMRT sequencing. The resulting assemblies are highly continuous, include fully resolved chromosome arms and close persistent gaps in these reference genomes. Our assembly of D. melanogaster revealed previously unknown heterochromatic and telomeric transition sequences, and we assembled low-complexity sequences from CHM1 that fill gaps in the human GRCh38 reference. Using MHAP and the Celera Assembler, single-molecule sequencing can produce de novo near-complete eukaryotic assemblies that are 99.99% accurate when compared with available reference genomes.


September 21, 2019  |  

A Sequel to Sanger: amplicon sequencing that scales.

Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system.By examining templates from more than 5000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion).SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year.


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