scholarly journals LAMBDR: Long-range amplification and Nanopore sequencing of the Mycobacterium bovis direct-repeat region. A novel method for in-silico spoligotyping of M. bovis directly from badger faeces

2019 ◽  
Author(s):  
R.S. James ◽  
E.R. Travis ◽  
A. D. Millard ◽  
P.C. Hewlett ◽  
L. Kravar-Garde ◽  
...  
Keyword(s):  
Long Range ◽  
Mycobacterium Bovis ◽  
Pcr Amplification ◽  
Direct Repeat ◽  
Repeat Region ◽  
Culture Independent ◽  
Long Read ◽  
Novel Method ◽  
Long Range Pcr ◽  
Template Dna

AbstractThe environment is an overlooked source of Mycobacterium bovis, the causative agent of bovine TB. Long read, end to end sequencing of variable repeat regions across the M. bovis genome was evaluated as a method of acquiring rapid strain level resolution directly from environmental samples. Eight samples of M. bovis, two BCG strains (Danish and Pasteur), and a single M. tuberculosis type culture (NCTC 13144) were used to generate data for this method. Long range PCR amplification of the direct repeat region was used to synthesize ∼5kb template DNA for onward sequence analysis. This has permitted culture independent identification of M. bovis spoligotypes present in the environment. Sequence level analysis of the direct repeat region showed that spoligotyping may underestimate strain diversity due to the inability to identify both SNPs and primer binding mutations using a biotinylated hybridisation approach.

scholarly journals Defining Blood Group Gene Reference Alleles by Long-Read Sequencing: Proof of Concept in the ACKR1 Gene Encoding the Duffy Antigens

2019 ◽  
Vol 47 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Yann Fichou ◽  
Isabelle Berlivet ◽  
Gaëlle Richard ◽  
Christophe Tournamille ◽  
Lilian Castilho ◽  
...  
Keyword(s):  
Blood Group ◽  
Single Molecule ◽  
Pcr Amplification ◽  
Null Alleles ◽  
Sequencing Technology ◽  
Gene Encoding ◽  
Next Generation Sequencing Technology ◽  
Sequencing Technologies ◽  
Long Read ◽  
Long Range Pcr

Background: In the novel era of blood group genomics, (re-)defining reference gene/allele sequences of blood group genes has become an important goal to achieve, both for diagnostic and research purposes. As novel potent sequencing technologies are available, we thought to investigate the variability encountered in the three most common alleles of ACKR1, the gene encoding the clinically relevant Duffy antigens, at the haplotype level by a long-read sequencing approach. Materials and Methods: After long-range PCR amplification spanning the whole ACKR1 gene locus (∼2.5 kilobases), amplicons generated from 81 samples with known genotypes were sequenced in a single read by using the Pacific Biosciences (PacBio) single molecule, real-time (SMRT) sequencing technology. Results: High-quality sequencing reads were obtained for the 162 alleles (accuracy >0.999). Twenty-two nucleotide variations reported in databases were identified, defining 19 haplotypes: four, eight, and seven haplotypes in 46 ACKR1*01, 63 ACKR1*02, and 53 ACKR1*02N.01 alleles, respectively. Discussion: Overall, we have defined a subset of reference alleles by third-generation (long-read) sequencing. This technology, which provides a “longitudinal” overview of the loci of interest (several thousand base pairs) and is complementary to the second-generation (short-read) next-generation sequencing technology, is of critical interest for resolving novel, rare, and null alleles.

scholarly journals Reflex™: a novel method to sequence barcoded long-range PCR products in a pooled population of hundreds of DNA samples

2012 ◽  
Vol 6 (S6) ◽  
Author(s):  
James Casbon ◽  
Andrew Slatter ◽  
Esther Musgrave-Brown ◽  
Robert Osborne ◽  
Conrad Lichtenstein ◽  
...  
Keyword(s):  
Long Range ◽  
Pcr Products ◽  
Novel Method ◽  
Long Range Pcr

LONG-RANGE PCR AMPLIFICATION AS AN ALTERNATIVE STRATEGY FOR CHARACTERIZING NOVEL HLA-B ALLELES

1996 ◽  
Vol 23 (4) ◽  
pp. 297-309 ◽  
Author(s):  
M. D. Curran ◽  
F. Williams ◽  
J. A. P. Earle ◽  
B. K. Rima ◽  
M. G. Dam ◽  
...  
Keyword(s):  
Long Range ◽  
Pcr Amplification ◽  
Alternative Strategy ◽  
Long Range Pcr

A combination of long-range PCR and long-read sequencing resolves common, high-risk dark and camouflaged carrier screening genes

2021 ◽  
Vol 132 ◽  
pp. S238-S239
Author(s):  
Sarah Statt ◽  
Julie Thibert ◽  
Jon Kemppainen ◽  
Cody Edwards ◽  
Liangjing Chen ◽  
...  
Keyword(s):  
High Risk ◽  
Long Range ◽  
Carrier Screening ◽  
Long Read ◽  
Long Range Pcr

scholarly journals Sequence Analysis of the Direct Repeat Region in Mycobacterium bovis

2001 ◽  
Vol 39 (3) ◽  
pp. 1067-1072 ◽  
Author(s):  
K. Caimi ◽  
M. I. Romano ◽  
A. Alito ◽  
M. Zumarraga ◽  
F. Bigi ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Mycobacterium Bovis ◽  
Direct Repeat ◽  
Repeat Region

scholarly journals Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

2021 ◽  
Vol 22 (4) ◽  
pp. 1508
Author(s):  
Jordi Maggi ◽  
Samuel Koller ◽  
Luzy Bähr ◽  
Silke Feil ◽  
Fatma Kivrak Pfiffner ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Long Range ◽  
Copy Number Variants ◽  
Retinal Disease ◽  
Promoter Regions ◽  
Long Range Pcr ◽  
Cost Efficient ◽  
Nucleotide Resolution ◽  
Genomic Regions ◽  
Next Generation Sequencing Ngs

The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

scholarly journals Long-read sequence confirmed a large deletion of MYH6 and MYH7 in a family with atrial septal defect

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
K Sonoda ◽  
S Ohno ◽  
M Horie
Keyword(s):  
Atrial Septal Defect ◽  
Septal Defect ◽  
Break Point ◽  
Large Deletion ◽  
Single Nucleotide Variants ◽  
Genomic Libraries ◽  
Flow Cells ◽  
Inherited Diseases ◽  
Long Read ◽  
Long Range Pcr

Abstract Background Genome structural variants (SVs) have larger effect on human genome functions than single nucleotide variants (SNVs). Although short-read sequencing (SRS) is current major next generation sequencing method and has given us a great benefit to elucidate the genetic background of inherited diseases, it does not detect SVs accurately. Long-read sequencing (LRS) produces tens to thousands of kilobases reads and detects the breakpoints of complex SVs. This study aimed to confirm a large deletion, which was suspected by SRS, using LRS by Oxford Nanopore technology (ONT). Methods Genomic libraries for SRS was prepared with HaloPlex. Targeted SRS was performed for 58 genes with MiSeq. Genomic libraries for LRS were prepared using the Ligation sequencing 1D kit SQK-LSK109 (ONT). Whole genome LRS was performed with GridION X5 and R9.4 flow cells (ONT). Results The patient was a five-month-old boy with atrial septal defect (ASD) and atrial tachycardia. Though SRS failed to identify any causative SNVs, the results with SureCall software (Agilent) suspected a deletion between exon 3 to exon 26 in MYH6 encoding α heavy chains of cardiac myosin. The variants in MYH6 are known to be associated with ASD. Because a deletion between MYH6 exon 26 and MYH7 exon 27 was reported as esv2748480 on the Database of Genomic Variants, we performed long-range PCR from MYH6 intron26 to MYH7 exon26 and found an abnormal 1.5K bases PCR product only in the case. Due to high homology of MYH6 and MYH7, Sanger sequencing failed to detect the break point. In LRS, 3 flow cells generated 3.8M base-called reads containing 42G bases with N50 of 13K bases. We used NGMLR, which is a long-read mapper, to align the reads to the human reference genome (hg38). SVs were called by Sniffles detecting all types of SVs. The deletion was found to range from chr14: 23390037 to 23419824 (see figure) and did not contain other SVs. There was no pathogenic SV on ACTC1, GATA4, TBX20 and TLL1 which are genes related to ASD on Genetic Testing Registry. His mother had also ASD and harbored the same deletion. Conclusions This is the first report to identify a large deletion between MYH6 and MYH7 in the family with ASD. The combination of SRS and LRS is useful to detect SVs in patients with suspected inherited diseases but carried no causative SNVs. Funding Acknowledgement Type of funding source: None

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