Accurate characterization of expanded tandem repeat length and sequence through whole genome long-read sequencing on PromethION

Abstract

De Roeck et al., PromethION sequencing and tandem repeat characterization 2 Abstract Tandem repeats (TRs) can cause disease through their length, sequence motif interruptions, and nucleotide modifications. For many TRs, however, these features are very difficult -if not impossible -to assess, requiring low-throughput and labor-intensive assays. One example is a VNTR in ABCA7 for which we recently discovered that expanded alleles strongly increase risk of Alzheimer's disease. Here, we investigated the potential of long-read whole genome sequencing to surmount these challenges, using the high-throughput PromethION platform from Oxford Nanopore Technologies. To overcome the limitations of conventional base calling and alignment, we developed an algorithm to study the TR size and sequence directly on raw PromethION current data.We report the long-read sequencing of multiple human genomes (n = 11) using only a single sequencing run and flow cell per individual. With the use of fresh DNA extractions, DNA shearing to approximately 20kb and size selection, we obtained an average output of 70 gigabases (Gb) per flow cell, corresponding to a 21x genome coverage, and a maximum yield of 98 Gb (30x genome coverage). All ABCA7 VNTR alleles, including expansions up to 10,000 bases, were spanned by long sequencing reads, validated by Southern blotting. Classical approaches of TR length estimation suffered from low accuracy, low precision, DNA strand effects and/or inability to call pathogenic repeat expansions. In contrast, our novel NanoSatellite algorithm, which circumvents base calling by using dynamic time warping on raw PromethION current data, achieved more than 90% accuracy and high precision (5.6% relative standard deviation) of TR length estimation, and detected all clinically relevant repeat expansions. In addition, we identified alternative TR sequence motifs with high consistency, allowing determination of TR sequence and distinction of VNTR alleles with homozygous length.In conclusion, we validated the robustness of single-experiment whole genome long-read sequencing on PromethION, a prerequisite for application of long-read sequencing in the clinic. In addition, we outperformed Southern blotting, enabling improved characterization of the role of expanded ABCA7 VNTR alleles in Alzheimer's disease, and opening new opportunities for TR research.

Targeted nanopore sequencing by real-time mapping of raw electrical signal with UNCALLED

“…Finally, we also intend to add an optional dynamic time warping (DTW) step to UNCALLED, making it a full-scale signal-to-basepair aligner. This could aid in raw signal applications outside of ReadUntil, such as assembly polishing, identifying nucleotide modifications 3 , and classifying variable number tandem repeats 37 . UNCALLED could improve the sensitivity of such analyses by eliminating the need for basecalling, which can be error prone around such features.…”

“…Deletions were characterized by first intersecting their reference coordinates with all RepeatMasker 28 entries downloaded from the UCSC Genome Browser 30 using bedtools 45 . If no overlap was found with RepeatMasker, we searched for overlaps with the "Simple Repeat" track from UCSC Genome Browser, which is based on Tandem Repeat Finder annotations 37 . Insertions were classified by extracting the insert sequence output by Sniffles for the PacBio HiFi SV calls, aligning this sequence to GRcH38 using BWA MEM 26 , and intersecting the alignments with RepeatMasker and Simple Repeats.…”

Structural variants identified by Oxford Nanopore PromethION sequencing of the human genome

“…Routine human genome sequencing has become possible on the recently commercially available PromethION sequencer. A PromethION flow cell has 3000 sensors and 12,000 pores, which generate on average 70 Gb of data in our hands, with a considerable variability (De Roeck et al 2018), allowing for the sequencing of a 20× covered human genome per flow cell. On PromethION devices, either 24 or 48 flow cells can be run simultaneously on the machine.…”

High-throughput multiplexed tandem repeat genotyping using targeted long-read sequencing

“…There have been few reports on the use of long-read sequencing for the analysis of specific TRs implicated in diseases [20–22]. Genotyping tools utilizing long-read sequencing data, such as Nanosatellite[21], RepeatHMM [23] and Tandem-genotypes [24] have been reported in the recent years with varying performance across different length of repeat units and repeat length. We reported VNTRTyper in Ganesamoorthy et.…”