10.1111/j.1399-0039.2010.01466.x PubMed Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. The post-sequencing bioinformatic post-processes are able to break down the CLR into shorter sub-reads, which encompass the sequence of one strand of the amplicon. 2020 Apr 23;15(4):e0232050. Four consensus sequences generated with SMRT DNA sequencing methods matched alleles that only had either partial gene or Coding DNA Sequences (CDS) available in the IMGT/HLA Database. The MHC sequencing consortium. Application of High-Throughput Next-Generation Sequencing for HLA Typing on Buccal Extracted DNA: Results from over 10,000 Donor Recruitment Samples. Data for the second HLA-B allele in sample AN3 initially suggested an intron 5 variant of HLA-B*27:05:02 (gDNA 2086C>T). Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. Much of the described polymorphism within the HLA class I genes is located within the exons that encode the peptide binding groove and the points at which T-cells interact with the molecule itself. All stages of the sequencing process, including library preparation, SMRT Cell loading and data collection were achieved within three working days. eCollection 2020. https://doi.org/10.1371/journal.pone.0127153.t002. Epub 2020 Feb 7. Eight novel genomic HLA class I sequences were identified, four were novel alleles, three were confirmed as genomic sequence extensions and one corrected an existing genomic reference sequence. Additionally rare or novel allele formed by a recombination event may be missed if the consensus sequence analysis tools are biased towards the more common alleles. The homozygous cell line chosen was the B-LCL COX (sample AN5) and was selected for HLA typing using the SMRT sequencing method as it is known to be consanguineous and has previously undergone in-depth sequence analysis of the entire Major Histocompatibility Complex (MHC) region on chromosome 6, which includes the HLA gene family [16, 17]. 1999;401(6756):921–3. Conceived and designed the experiments: NPM JR AJMM SR KE C-SC HB JAM KL SGEM. Nature. The amplicon lengths were similar enough to negate the potential problem of loading bias towards smaller PCR products in a pool when dispensed into SMRT Cells. Written consent for HLA typing was obtained from all blood and saliva sample donors at the point of collection for the purposes of transplantation. The down-stream uses of HLA typing data are varied and include assessing compatibility between donors and recipients prior to transplantation, drug hypersensitivity and disease associations. In order to assess the accuracy of SMRT DNA sequencing technology, we determined the number of homopolymer regions present in each of the 38 HLA sequences generated that consisted of five or more nucleotides (Table 5). 2014. Seven DNA samples were selected for HLA class I genotyping using Pacific Biosciences’ SMRT sequencing methodology. 2010;75(4):291–455. 2020 Apr 23;15(4):e0232050. doi: 10.1371/journal.pone.0165810. NCI CPTC Antibody Characterization Program, Horton R, Wilming L, Rand V, Lovering RC, Bruford EA, Khodiyar VK, et al. In combination, these factors make the SMRT DNA sequencing method amenable for use in a high-throughput HLA typing laboratory. The ideal solution to resolve both HLA ambiguity and the potential problems caused by phasing multiple fragments would be to produce multiple long sequence reads encompassing whole gene PCR amplicons, in isolation. Anthony Nolan Research Institute, Royal Free Hospital, London, United Kingdom, 2016 Oct 31;11(10):e0165810. Get the latest research from NIH: https://www.nih.gov/coronavirus. Sequences corresponding to the HLA pseudogene allele HLA-H*02:01:01:01 were identified, although the numbers of reads were low (n = 12). It is important that larger and more diverse cohorts of DNA samples are sequenced using SMRT DNA technology to confirm suitability for HLA typing. Fig 1. Epub 2019 Dec 11. Robinson J, Barker DJ, Georgiou X, Cooper MA, Flicek P, Marsh SGE. Unexpectedly, an additional HLA gene sequence was also identified in this sample. As all amplicons in this experiment were of concentrations towards the upper limits of the kit, it is possible that the sizing and quantification values were affected, which consequently affected the volumes required for equimolar pooling and causing the imbalance between loci. No, PLOS is a nonprofit 501(c)(3) corporation, #C2354500, based in San Francisco, California, US, https://doi.org/10.1371/journal.pone.0127153, https://doi.org/10.1016/j.humimm.2012.12.007. Seven DNA samples were typed for HLA-A, -B and -C. The results showed that SMRT DNA sequencing technology was able to generate sequences that spanned entire HLA Class I genes that allowed for accurate allele calling. Clipboard, Search History, and several other advanced features are temporarily unavailable. Liu C, Duffy BF, Weimer ET, Montgomery MC, Jennemann JE, Hill R, Phelan D, Lay L, Parikh BA. Get the latest public health information from CDC: https://www.coronavirus.gov. 2020 Apr;47(2):139-148. doi: 10.1111/iji.12479. Noticeably fewer homopolymer regions were observed for the A, C and G nucleotides, particularly in 7-, 8- and 9-mers. Epub 2010/04/02.  |  The function of these adaptors is to turn an essentially linear PCR amplicon into a circular molecule. Of particular interest was the accuracy of the data produced for the homopolymer regions present in the different alleles due to the known cross-platform problem of enzymes incurring slippage when sequencing through long stretches of a single continuous base.

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