De Novo assembly

Long sequencing reads (several tens of kb) greatly facilitate de novo genome assembly and variants phasing. It also allows the analysis of structural variants (insertions, deletions, repetitions of regions of several tens of nucleotides) which are invisible with short read sequencing data.

De novo assembly has successfully been applied to bacteria, insects, plants, mammals, and more.

De novo human genome assembly of HiFi requires less computing than short reads or non HiFi long reads (typically less than 1-day computing on a standard system).

Variant detection

HiFi reads length, accuracy, and uniformity open access to genomic regions impossible to analyze with short reads sequencing.

Table shows improvement in mappability with 13.5 kb HiFi reads for 193 human genes previously reported as medically relevant and problematic to map with NGS reads. The lower panel shows coverage of the STRC gene with either short reads (2×151 bp) or 13.5 kb HiFi reads. Source Wenger et al. Nature Biotechnology, 2019

The Truth Challenge V2 from the FDA, aimed at comparing technologies for calling variants in difficult-to-map regions, revealed that HiFi reads can outperform short read sequencing.

Full-Length RNA sequencing

For Pacific Biosciences sequencing, full-Length RNA sequencing (from 5′ cap to 3′ poly A tail) is called ISOseq. The high-quality sequences obtained give a qualitative view of transcript isoform diversity, even for very short exons.

Source: Pacific Biosciences Technote: Why is full length RNA sequencing useful?

The sequencing saturation offered by PacBio sequencing is so far not sufficient for standard transcriptome profiling. It is therefore a qualitative approach only. Quantitative approaches are under development (Pacific Biosciences and Oxford Nanopore) but this is not fully supported by GTF yet.

The main applications are:
Genome annotation
New gene, or transcript isoform discovery

Along the same line, methods are being developed for performing ISOseq at the single cell level (scISOseq). The double stranded full-length cDNA, amplified as part of the 10X genomics single cell RNAseq procedure, is the template for ISOseq library preparation.

Typical structure of a single cell derived 10X full length double strand cDNA

Amplicon sequencing

Another benefit of long read sequencing, of particular interest for amplicon sequencing, lies in the fact that variants can easily be phased (see below). This information is key for applications such as HLA typing of VDJ sequencing for instance.

HiFi reads (long and accurate) allow variant phasing

Full length 16S sequencing is another key application for high accuracy long read amplicon sequencing. Getting access to the full 1.5 kb 16S sequence indeed greatly improves sensitivity and precision compared to standard short variable region analysis (see figure below), potentially “providing taxonomic resolution of bacterial communities at species and strain level”.

Modified from Johnson et al., Nat. Communication, 2019 [16S database = GREENGENES]