Genetic Findings of Sanger and Nanopore Single-Molecule Sequencing in Patients with X-Linked Hearing Loss and Incomplete Partition Type III

Abstract BackgroundPOU3F4 is the causative gene for X-linked deafness-2 (DFNX2), characterized by incomplete partition type III (IP-III) malformation of the inner ear. The aim of this study was to investigate the clinical characteristics and molecular findings by Sanger or Nanopore single-molecule sequencing in IP-III patients. MethodsDiagnosis of IP-III was mainly based on clinical characteristics including radiological and audiological findings. Sanger sequencing of POU3F4 were carried out for these IP-III patients. For those patients with negative results for POU3F4 Sanger sequencing, Nanopore long-read single-molecule sequencing was used to identify the possible pathogenic variants. Hearing intervention outcomes of hearing aids fitting and cochlear implantation were also analyzed. Grouped by different locations of POU3F4 variants, aided PTA was further compared between patients in whom the variants located in the exon region or in the upstream region.ResultsIn total, 18 male patients from 14 unrelated families were diagnosed with IP-III. 10 variants were identified in POU3F4 by Sanger sequencing and 9 of these were novel (p.Val321Gly, p.Gln181*, p.Cys233*, p.Val215Gly, p.Arg282Gln, p.Trp57*, p.Gln316*, c.903_912 delins TGCCA and p.Arg205del). Four different deletions (DELs) that varied from 80 to 486 kb were identified 876-1503 kb upstream of POU3F4 by Nanopore long-read single-molecule sequencing. Of them, de novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutations. Of these 18 patients, 7 had bilateral hearing aids (HAs) and 10 patients received unilateral cochlear implantation (CI). The mean aided pure tone average (PTA) for HAs and CI users were 41.1±5.18 and 40.3±7.59 dB HL respectively. The mean PTAs for whom the variants located in the exon and upstream regions were 39.6±6.31 vs 43.0±7.10 dB HL, which presented no significant difference (p=0.342).ConclusionsAmong IP-III patients, 28.6% (4/14) had no definite mutation in exon region of POU3F4, however, possible pathogenic deletions were identified in upstream region of this gen. De novo genetic mutations occurred in 21.4% (3/14) of patients with POU3F4 mutation. Hearing intervention outcomes of IP-III patients presented no difference regardless of the variants locations on exon or upstream regions.

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A whole genome atlas of 81 Psilocybe genomes as a resource for psilocybin production.

F1000Research ◽

10.12688/f1000research.55301.2 ◽

2021 ◽

Vol 10 ◽

pp. 961

Author(s):

Kevin McKernan ◽

Liam Kane ◽

Yvonne Helbert ◽

Lei Zhang ◽

Nathan Houde ◽

...

Keyword(s):

Gene Cluster ◽

Single Molecule ◽

Reference Genome ◽

De Novo ◽

Genomic Diversity ◽

Sequence Coverage ◽

Single Molecule Sequencing ◽

Contiguous Gene ◽

Long Read ◽

Interesting Variation

The Psilocybe genus is well known for the synthesis of valuable psychoactive compounds such as Psilocybin, Psilocin, Baeocystin and Aeruginascin. The ubiquity of Psilocybin synthesis in Psilocybe has been attributed to a horizontal gene transfer mechanism of a ~20Kb gene cluster. A recently published highly contiguous reference genome derived from long read single molecule sequencing has underscored interesting variation in this Psilocybin synthesis gene cluster. This reference genome has also enabled the shotgun sequencing of spores from many Psilocybe strains to better catalog the genomic diversity in the Psilocybin synthesis pathway. Here we present the de novo assembly of 81 Psilocybe genomes compared to the P.envy reference genome. Surprisingly, the genomes of Psilocybe galindoi, Psilocybe tampanensis and Psilocybe azurescens lack sequence coverage over the previously described Psilocybin synthesis pathway but do demonstrate amino acid sequence homology to a less contiguous gene cluster and may illuminate the previously proposed evolution of psilocybin synthesis.

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AsmMix: A pipeline for high quality diploid de novo assembly

10.1101/2021.01.15.426893 ◽

2021 ◽

Author(s):

Pei Wu ◽

Chao Liu ◽

Ou Wang ◽

Xia Zhao ◽

Fang Chen ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Variant Calling ◽

The Other ◽

Second Step ◽

Small Scale ◽

Mixing Process ◽

High Quality ◽

Single Molecule Sequencing ◽

Long Read

AbstractIn this paper, we report a pipeline, AsmMix, which is capable of producing both contiguous and high-quality diploid genomes. The pipeline consists of two steps. In the first step, two sets of assemblies are generated: one is based on co-barcoded reads, which are highly accurate and haplotype-resolved but contain many gaps, the other assembly is based on single-molecule sequencing reads, which is contiguous but error-prone. In the second step, those two sets of assemblies are compared and integrated into a haplotype-resolved assembly with fewer errors. We test our pipeline using a dataset of human genome NA24385, perform variant calling from those assemblies and then compare against GIAB Benchmark. We show that AsmMix pipeline could produce highly contiguous, accurate, and haplotype-resolved assemblies. Especially the assembly mixing process could effectively reduce small-scale errors in the long read assembly.

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Assembling Large Genomes with Single-Molecule Sequencing and Locality Sensitive Hashing

10.1101/008003 ◽

2014 ◽

Cited By ~ 13

Author(s):

Konstantin Berlin ◽

Sergey Koren ◽

Chen-Shan Chin ◽

James Drake ◽

Jane M Landolin ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Locality Sensitive Hashing ◽

Model Organisms ◽

Smrt Sequencing ◽

High Coverage ◽

Celera Assembler ◽

Single Molecule Sequencing ◽

Long Reads ◽

Long Read

We report reference-grade de novo assemblies of four model organisms and the human genome from single-molecule, real-time (SMRT) sequencing. Long-read SMRT sequencing is routinely used to finish microbial genomes, but the available assembly methods have not scaled well to larger genomes. Here we introduce the MinHash Alignment Process (MHAP) for efficient overlapping of noisy, long reads using probabilistic, locality-sensitive hashing. Together with Celera Assembler, MHAP was used to reconstruct the genomes of Escherichia coli, Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, and human from high-coverage SMRT sequencing. The resulting assemblies include fully resolved chromosome arms and close persistent gaps in these important reference genomes, including heterochromatic and telomeric transition sequences. For D. melanogaster, MHAP achieved a 600-fold speedup relative to prior methods and a cloud computing cost of a few hundred dollars. These results demonstrate that single-molecule sequencing alone can produce near-complete eukaryotic genomes at modest cost.

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lra: A long read aligner for sequences and contigs

PLoS Computational Biology ◽

10.1371/journal.pcbi.1009078 ◽

2021 ◽

Vol 17 (6) ◽

pp. e1009078

Author(s):

Jingwen Ren ◽

Mark J. P. Chaisson

Keyword(s):

Dynamic Programming ◽

Single Molecule ◽

De Novo Assembly ◽

De Novo ◽

Concave Function ◽

Single Molecule Sequencing ◽

Link Type ◽

Oxford Nanopore ◽

Concave Cost ◽

Long Read

It is computationally challenging to detect variation by aligning single-molecule sequencing (SMS) reads, or contigs from SMS assemblies. One approach to efficiently align SMS reads is sparse dynamic programming (SDP), where optimal chains of exact matches are found between the sequence and the genome. While straightforward implementations of SDP penalize gaps with a cost that is a linear function of gap length, biological variation is more accurately represented when gap cost is a concave function of gap length. We have developed a method, lra, that uses SDP with a concave-cost gap penalty, and used lra to align long-read sequences from PacBio and Oxford Nanopore (ONT) instruments as well as de novo assembly contigs. This alignment approach increases sensitivity and specificity for SV discovery, particularly for variants above 1kb and when discovering variation from ONT reads, while having runtime that are comparable (1.05-3.76×) to current methods. When applied to calling variation from de novo assembly contigs, there is a 3.2% increase in Truvari F1 score compared to minimap2+htsbox. lra is available in bioconda (https://anaconda.org/bioconda/lra) and github (https://github.com/ChaissonLab/LRA).

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Single-molecule sequencing of theDrosophila serratagenome

10.1101/090969 ◽

2016 ◽

Author(s):

Scott L. Allen ◽

Emily K. Delaney ◽

Artyom Kopp ◽

Stephen F. Chenoweth

Keyword(s):

Single Molecule ◽

De Novo ◽

Sequence Data ◽

Error Rates ◽

Model Species ◽

High Coverage ◽

Single Molecule Sequencing ◽

Long Read ◽

Drosophila Serrata ◽

High Degree

ABSTRACTLong read sequencing technology promises to greatly enhancede novoassembly of genomes for non-model species. While error rates have been a large stumbling block, sequencing at high coverage allows reads to be self-corrected. Here we sequence andde novoassemble the genome ofDrosophila serrata, a non-model species from themontiumsubgroup that has been well studied for clines and sexual selection. Using 11 PacBio SMRT cells, we generated 12 Gbp of raw sequence data comprising approximately 65x whole genome coverage. Read lengths averaged 8,940 bp (NRead50 12,200) with the longest read at 53 Kbp. We self-corrected reads using the PBDagCon algorithm and assembled the genome using the MHAP algorithm within the PBcR assembler. Total genome length was 198 Mbp with an N50 just under 1 Mbp. Contigs displayed a high degree of arm-level conservation withD. melanogaster. We also provide an initial annotation for this genome usingin silicogene predictions that were supported by RNA-seq data.

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lra: the Long Read Aligner for Sequences and Contigs

10.1101/2020.11.15.383273 ◽

2020 ◽

Author(s):

Jingwen Ren ◽

Mark JP Chaisson

Keyword(s):

Single Molecule ◽

De Novo ◽

Data Types ◽

Single Molecule Sequencing ◽

Detection Algorithms ◽

Link Type ◽

Long Reads ◽

Oxford Nanopore ◽

Long Read ◽

Linear Cost

AbstractMotivationIt is computationally challenging to detect variation by aligning long reads from single-molecule sequencing (SMS) instruments, or megabase-scale contigs from SMS assemblies. One approach to efficiently align long sequences is sparse dynamic programming (SDP), where exact matches are found between the sequence and the genome, and optimal chains of matches are found representing a rough alignment. Sequence variation is more accurately modeled when alignments are scored with a gap penalty that is a convex function of the gap length. Because previous implementations of SDP used a linear-cost gap function that does not accurately model variation, and implementations of alignment that have a convex gap penalty are either inefficient or use heuristics, we developed a method, lra, that uses SDP with a convex-cost gap penalty. We use lra to align long-read sequences from PacBio and Oxford Nanopore (ONT) instruments as well as de novo assembly contigs.ResultsAcross all data types, the runtime of lra is between 52-168% of the state of the art aligner minimap2 when generating SAM alignment, and 9-15% of an alternative method, ngmlr. This alignment approach may be used to provide additional evidence of SV calls in PacBio datasets, and an increase in sensitivity and specificity on ONT data with current SV detection algorithms. The number of calls discovered using pbsv with lra alignments are within 98.3-98.6% of calls made from minimap2 alignments on the same data, and give a nominal 0.2-0.4% increase in F1 score by Truvari analysis. On ONT data with SV called using Sniffles, the number of calls made from lra alignments is 3% greater than minimap2-based calls, and 30% greater than ngmlr based calls, with a 4.6-5.5% increase in Truvari F1 score. When applied to calling variation from de novo assembly contigs, there is a 5.8% increase in SV calls compared to minimap2+paftools, with a 4.3% increase in Truvari F1 score.Availability and implementationAvailable in bioconda: https://anaconda.org/bioconda/lra and github: https://github.com/ChaissonLab/[email protected], [email protected]

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SMARTdenovo: a de novo assembler using long noisy reads

Gigabyte ◽

10.46471/gigabyte.15 ◽

2021 ◽

Vol 2021 ◽

pp. 1-9

Author(s):

Hailin Liu ◽

Shigang Wu ◽

Alun Li ◽

Jue Ruan

Keyword(s):

Error Correction ◽

Single Molecule ◽

Genome Assembly ◽

De Novo ◽

Structural Variants ◽

High Quality ◽

Single Molecule Sequencing ◽

Long Read ◽

Reference Quality

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. It has also been widely used to study structural variants, phase haplotypes and more. Here, we introduce the assembler SMARTdenovo, a single-molecule sequencing (SMS) assembler that follows the overlap-layout-consensus (OLC) paradigm. SMARTdenovo (RRID: SCR_017622) was designed to be a rapid assembler, which, unlike contemporaneous SMS assemblers, does not require highly accurate raw reads for error correction. It has performed well in the evaluation of congeneric assemblers and has been successfully users for various assembly projects. It is compatible with Canu for assembling high-quality genomes, and several of the assembly strategies in this program have been incorporated into subsequent popular assemblers. The assembler has been in use since 2015; here we provide information on the development of SMARTdenovo and how to implement its algorithms into current projects.

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SMARTdenovo: A de novo Assembler Using Long Noisy Reads

10.20944/preprints202009.0207.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Hailin Liu ◽

Shigang Wu ◽

Alun Li ◽

Jue Ruan

Keyword(s):

Error Correction ◽

Single Molecule ◽

Genome Assembly ◽

De Novo ◽

Structural Variants ◽

High Quality ◽

De Novo Genome Assembly ◽

Single Molecule Sequencing ◽

Long Read ◽

Reference Quality

Long-read single-molecule sequencing has revolutionized de novo genome assembly and enabled the automated reconstruction of reference-quality genomes. It also has been widely used to study structural variants, phase haplotypes and more. Here, we introduce the assembler— SMARTdenovo, which is an SMS assembler that follows the overlap-layout-consensus (OLC) paradigm. SMARTdenovo (RRID: SCR_017622) was designed to be a fast assembler that did not require highly accurate raw reads for error correction, unlike other, contemporaneous SMS assemblers. It has performed well for evaluating congeneric assemblers and has been successful for a variety of assembly projects. It is compatible with Canu for assembling high-quality genomes, and several of the assembly strategies in this program have been incorporated into subsequent popular assemblers. The assembler has been in use since 2015, and here we provide information on the development of SMARTdenovo and how to implement its algorithms into current projects.

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A whole genome atlas of 81 Psilocybe genomes as a resource for psilocybin production.

F1000Research ◽

10.12688/f1000research.55301.1 ◽

2021 ◽

Vol 10 ◽

pp. 961

Author(s):

Kevin McKernan ◽

Liam Kane ◽

Yvonne Helbert ◽

Lei Zhang ◽

Nathan Houde ◽

...

Keyword(s):

Single Molecule ◽

Reference Genome ◽

De Novo ◽

Alternative Pathway ◽

Gene Cassette ◽

Genomic Diversity ◽

Sequence Coverage ◽

Single Molecule Sequencing ◽

Long Read ◽

Interesting Variation

The Psilocybe genus is well known for the synthesis of valuable psychoactive compounds such as Psilocybin, Psilocin, Baeocystin and Aeruginascin. The ubiquity of Psilocybin synthesis in Psilocybe has been attributed to a horizontal gene transfer mechanism of a ~20Kb gene cassette. A recently published highly contiguous reference genome derived from long read single molecule sequencing has underscored interesting variation in this Psilocybin synthesis gene cassette. This reference genome has also enabled the shotgun sequencing of spores from many Psilocybe strains to better catalog the genomic diversity in the Psilocybin synthesis pathway. Here we present the de novo assembly of genomes of 81 Psilocybe genomes compared to the P.envy reference genome. Surprisingly, the genomes of Psilocybe galindoi, Psilocybe tampanensis and Psilocybe azurescens lack sequence coverage over the previously described Psilocybin synthesis pathway but do demonstrate amino acid sequence homology to an alternative pathway and may illuminate previously proposed convergent evolution of Psilocybin synthesis.

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Mapping and phasing of structural variation in patient genomes using nanopore sequencing

10.1101/129379 ◽

2017 ◽

Cited By ~ 4

Author(s):

Mircea Cretu Stancu ◽

Markus J. van Roosmalen ◽

Ivo Renkens ◽

Marleen Nieboer ◽

Sjors Middelkamp ◽

...

Keyword(s):

Single Molecule ◽

De Novo ◽

Structural Variants ◽

Human Genetic Disease ◽

Structural Genomic ◽

Short Read ◽

Sequencing Technologies ◽

Genome Wide ◽

Long Read ◽

Complex Structural

AbstractStructural genomic variants form a common type of genetic alteration underlying human genetic disease and phenotypic variation. Despite major improvements in genome sequencing technology and data analysis, the detection of structural variants still poses challenges, particularly when variants are of high complexity. Emerging long-read single-molecule sequencing technologies provide new opportunities for detection of structural variants. Here, we demonstrate sequencing of the genomes of two patients with congenital abnormalities using the ONT MinION at 11x and 16x mean coverage, respectively. We developed a bioinformatic pipeline - NanoSV - to efficiently map genomic structural variants (SVs) from the long-read data. We demonstrate that the nanopore data are superior to corresponding short-read data with regard to detection of de novo rearrangements originating from complex chromothripsis events in the patients. Additionally, genome-wide surveillance of SVs, revealed 3,253 (33%) novel variants that were missed in short-read data of the same sample, the majority of which are duplications < 200bp in size. Long sequencing reads enabled efficient phasing of genetic variations, allowing the construction of genome-wide maps of phased SVs and SNVs. We employed read-based phasing to show that all de novo chromothripsis breakpoints occurred on paternal chromosomes and we resolved the long-range structure of the chromothripsis. This work demonstrates the value of long-read sequencing for screening whole genomes of patients for complex structural variants.

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