Auto Tag: Anthony Nolan

June 1, 2021  |  

Genomic DNA sequences of HLA class I alleles generated using multiplexed barcodes and SMRT DNA Sequencing technology.

Allelic-level resolution HLA typing is known to improve survival prognoses post Unrelated Donor (UD) Haematopoietic Stem Cell Transplantation (HSCT). Currently, many commonly used HLA typing methodologies are limited either due to the fact that ambiguity cannot be resolved or that they are not amenable to high-throughput laboratories. Pacific Biosciences’ Single Molecule Real-Time (SMRT) DNA sequencing technology enables sequencing of single molecules in isolation and has read-length capabilities to enable whole gene sequencing for HLA. DNA barcode technology labels samples with unique identifiers that can be traced throughout the sequencing process. The use of DNA barcodes means that multiple samples can be sequenced in a single experiment but data can still be attributed to the correct sample. Here we describe the results of experiments that use DNA barcodes to facilitate sequencing of multiple samples for full-length HLA class I genes (known as multiplexing).

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September 22, 2019  |  

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.

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September 22, 2019  |  

Report from the Killer-cell Immunoglobulin-like Receptors (KIR) component of the 17th International HLA and Immunogenetics Workshop.

The goals of the KIR component of the 17th International HLA and Immunogenetics Workshop (IHIW) were to encourage and educate researchers to begin analyzing KIR at allelic resolution, and to survey the nature and extent of KIR allelic diversity across human populations. To represent worldwide diversity, we analyzed 1269 individuals from ten populations, focusing on the most polymorphic KIR genes, which express receptors having three immunoglobulin (Ig)-like domains (KIR3DL1/S1, KIR3DL2 and KIR3DL3). We identified 13 novel alleles of KIR3DL1/S1, 13 of KIR3DL2 and 18 of KIR3DL3. Previously identified alleles, corresponding to 33 alleles of KIR3DL1/S1, 38 of KIR3DL2, and 43 of KIR3DL3, represented over 90% of the observed allele frequencies for these genes. In total we observed 37 KIR3DL1/S1 allotypes, 40 for KIR3DL2 and 44 for KIR3DL3. As KIR allotype diversity can affect NK cell function, this demonstrates potential for high functional diversity worldwide. Allelic variation further diversifies KIR haplotypes. We determined KIR3DL3?~?KIR3DL1/S1?~?KIR3DL2 haplotypes from five of the studied populations, and observed multiple population-specific haplotypes in each. This included 234 distinct haplotypes in European Americans, 191 in Ugandans, 35 in Papuans, 95 in Egyptians and 86 in Spanish populations. For another 35 populations, encompassing 642,105 individuals we focused on KIR3DL2 and identified another 375 novel alleles, with approximately half of them observed in more than one individual. The KIR allelic level data gathered from this project represents the most comprehensive summary of global KIR allelic diversity to date, and continued analysis will improve understanding of KIR allelic polymorphism in global populations. Further, the wealth of new data gathered in the course of this workshop component highlights the value of collaborative, community-based efforts in immunogenetics research, exemplified by the IHIW.Copyright © 2018. Published by Elsevier Inc.

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