A Long-Read Sequencing Approach for Direct Haplotype Phasing in Clinical Settings

Abstract

The reconstruction of individual haplotypes can facilitate the interpretation of disease risks; however, high costs and technical challenges still hinder their assessment in clinical settings. Second-generation sequencing is the gold standard for variant discovery but, due to the production of short reads covering small genomic regions, allows only indirect haplotyping based on statistical methods. In contrast, third-generation methods such as the nanopore sequencing platform developed by Oxford Nanopore Technologies (ONT) generate long reads that can be used for direct haplotyping, with fewer drawbacks. However, robust standards for variant phasing in ONT-based target resequencing efforts are not yet available. In this study, we presented a streamlined proof-of-concept workflow for variant calling and phasing based on ONT data in a clinically relevant 12-kb region of the APOE locus, a hotspot for variants and haplotypes associated with aging-related diseases and longevity. Starting with sequencing data from simple amplicons of the target locus, we demonstrated that ONT data allow for reliable single-nucleotide variant (SNV) calling and phasing from as little as 60 reads, although the recognition of indels is less efficient. Even so, we identified the best combination of ONT read sets (600) and software (BWA/Minimap2 and HapCUT2) that enables full haplotype reconstruction when both SNVs and indels have been identified previously using a highly-accurate sequencing platform. In conclusion, we established a rapid and inexpensive workflow for variant phasing based on ONT long reads. This allowed for the analysis of multiple samples in parallel and can easily be implemented in routine clinical practice, including diagnostic testing.

Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture

“…Xdrop allowed the enrichment of a genomic region containing the entire FMR1 gene, so we next analyzed intragenic SNVs and indels in the same four samples discussed above ( Supplementary Table S1 ). At this aim, we exploited Illumina sequencing ( Supplementary Table S1 ), because with ONT most of gene body (74%) had coverage <60X ( Figure 1F ), i.e., lower than the minimum threshold required to accurately call SNV using this technology ( Maestri et al, 2020 ). Analysis of the five distinct dMDA replicates of the NA12878 sample, for which genotypes are available, demonstrated most of the GIAB variants were properly called by each replicate, with 93% sensitivity on average ( Supplementary Table S2 ).…”

“…Finally, the system lacks flexibility, because commonly utilized protocols to sequence the Cas9-enriched DNA rely only on long-read sequencing and not on short-read sequencing platforms, such as Illumina, which show higher accuracy. Despite recent improvements strongly increased long-read accuracy, ONT still fails at accurately detecting indels ( Maestri et al, 2020 ), while PacBio High-Fidelity mode still requires the use of high-capacity SMRT cells, that makes the analysis very expensive when only few samples are multiplexed. These are critical drawbacks, especially when the repeat characterization is inconclusive and the analysis of the entire gene is necessary to identify other mutations, namely, SNVs or indels ( Sitzmann et al, 2018 ).…”

Features and applications of haplotypes in crop breeding

“…However, the availability of the high-throughput sequencing platforms has made a tremendous impact on the identification of haplotypes and their application in the genetic studies. Although, the second-generation sequencing techniques produce short reads of 150 bp, these small reads normally do not possess more than a single variant 88 . Hence, the haplotypes are constructed indirectly from this data and this needs specific statistical inferences from population genotyping data, which in turn increases the time and cost for the haplotype construction 88 , 89 .…”

“…Although, the second-generation sequencing techniques produce short reads of 150 bp, these small reads normally do not possess more than a single variant 88 . Hence, the haplotypes are constructed indirectly from this data and this needs specific statistical inferences from population genotyping data, which in turn increases the time and cost for the haplotype construction 88 , 89 . In contrast, third-generation sequencing (TGS), such as the Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT), produce long reads from which the haplotypes can be directly constructed 88 .…”

The application of Nanopore sequencing for variant calling on the human mitochondrial DNA

“…At the same time, long reads have found application in revealing structural variants (Aganezov et al, 2020), phasing analysis with haplotype reconstruction (Maestri et al, 2020;Popitsch et al, 2020), and the nanoporebased assay for analyzing leukemic samples (Orsini et al, 2018;Cumbo et al, 2019). Nevertheless, reports on long reads' usage with regard to mitochondrial genetics are still scarce.…”