Improved de novo assembly with nanopore ultra-long and duplex data, and scaffolding using Pore-C

We used 60x of nanopore ultra-long reads (read N50 > 100 kb) base called with guppy 5 (SUP model) to produce a highly contiguous assembly of Hg002 using Flye 2.9. The final assembly has a contig N50 of 62 Mb (Fig. 1a). The largest contig was 196 Mb and 90% of the genome was contained in contigs > 15 Mb. The assembly showed high accuracy, yielding a BUSCO score of 96.9% (complete genes). Fig. 1b shows contig sizes along the chromosomes. Colour changes between beige and blue show contig or alignment breaks. For half the chromosomes, >95% of sequence assembled into 5 fragments or fewer. The efficiency of long-read assembly and polishing tools means that the overall runtime was <72 hours on a single AWS instance.

To demonstrate the effectiveness of scaffolding assemblies with Pore-C data, we performed de novo assembly using Flye for all genomes apart from human (NA12878), which we assembled with Shasta. For each genome, Pore-C data was processed to create virtual paired contacts, which were used for scaffolding the assemblies. The results show that scaffolding with approximately 10x Pore-C data can increase assembly contiguity substantially, even when the initial draft assembly is highly fragmented (Figs. 2a-2d). Where scaffolded assembly N50 is greater than the reference, it indicates that sequence that is missing from the current reference assemblies.

Oxford Nanopore’s duplex pipeline finds reads originating from both strands of the same double-stranded DNA molecule by comparing reads which pass through the same pore in succession. Duplex base-calling combines signal information from both strands and can generate a 2-pass consensus read sequence with >99.9% modal accuracy (>30 on the Phred scale). Read accuracy from duplex base-calling is independent of read length (Figs. 3a and 3b, top), allowing for arbitrarily long highly accurate reads. These reads can be assembled with the latest generation of high-performance genome assembly tools, including Hifiasm, HiCanu, Verkko (Figs. 3a and 3b, bottom), and La Jolla Assembler (Fig. 3c), leading to near complete chromosome or chromosome-arm on single contigs (Figs. 3a and 3b, bottom). Longer read lengths unlock higher-contiguity assemblies even at low coverages, as can be seen in our assemblies of different length 20x subsets of C. elegans duplex data; assembly of 20x of duplex reads longer than 30 kb (read length N50 = 41 kb) led to an assembly with a 25% higher contig N50 compared with 20x of duplex reads between 15-30 kb (read length N50 = 22 kb) and an order of magnitude higher contig N50 compared with 20x of duplex reads between 5-15 kb (read length N50 = 10 kb). High accuracy reads also significantly improve assembly of samples for which obtaining high molecular weight DNA continues to be challenging, as in some bacteria. For example, a 50-200x duplex dataset for the Zymo mock community with read N50s of ca. 5 kb was mostly assembled to single, nearly perfect circular contigs by the MetaFlye assembler (Fig. 3d).