Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion

Zhang, Zhe; An, Hyun Hyung; Vege, Sunitha; Hu, Tai‐Shan; Zhang, Shiping; Mosbruger, Timothy; Jayaraman, Padmini; Monos, Dimitri; Westhoff, Connie M.; Chou, Stella T.

doi:10.1016/j.ajhg.2021.12.003

Cited by 20 publications

(14 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Recently, long-read sequencing technologies for generating continuous sequences of a single molecule have been developed 9 and their application to the blood group genes has been reported. [10][11][12][13][14] The long-read and accuracy of HiFi reads enabled us to determine the 20 Kb-long sequence, including the initiation and termination codons, of the JK gene. In addition, we obtained more than 750 HiFi-reads of the JK silencing alleles (data not shown); however, 30 randomly selected reads were sufficient to determine the sequences in the present study.…”

Section: Discussionmentioning

confidence: 99%

“…In terms of genomic DNA analysis using Sanger sequencing or short‐read‐based next generation sequencing, it is difficult to determine the cis or trans positions of SNVs, which are occasionally more than 1 Kb away from each other. Recently, long‐read sequencing technologies for generating continuous sequences of a single molecule have been developed 9 and their application to the blood group genes has been reported 10–14 . The long‐read and accuracy of HiFi reads enabled us to determine the 20 Kb‐long sequence, including the initiation and termination codons, of the JK gene.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

et al. 2023

View full text Add to dashboard Cite

BackgroundThe Kidd blood group gene SLC14A1 (JK) accounts for approximately 20 Kb from initiation codon to stop codon in the genome. In genomic DNA analysis using Sanger sequencing or short‐read‐based next generation sequencing, it is difficult to determine the cis or trans positions of single nucleotide variations (SNVs), which are occasionally more than 1 Kb away from each other. We aimed to determine the complete nucleotide sequence of a 20‐Kb genomic DNA amplicon to characterize the JK allelic variants associated with Kidd antigen silencing in a blood donor.Study Design and MethodsThe Jk(a–b–) phenotype was identified in this donor by standard serological typing. A DNA sample obtained from whole blood was amplified by long‐range PCR to obtain a 20‐Kb fragment of the SLC14A1 gene, including the initiation and stop codons. The fragment was then analyzed by Sanger sequencing and single‐molecule sequencing. Transfection and expression studies were performed in CHO cells using the expression vector construct of JK alleles.ResultsSanger sequencing and single‐molecule sequencing revealed that the donor was heterozygous with JK*01 having c.276G>A (rs763262711, p.Trp92Ter) and JK*02 having c.499A>G (rs2298719, p.Met167Val), c.588A>G (rs2298718, p.Pro196Pro), and c.743C>A (p.Ala248Asp). The two JK alleles identified have not been previously described. Transfection and expression studies indicated that the CHO cells transfected with JK*02 having c.743C>A did not express the Jkb and Jk3 antigens.ConclusionsWe identified new JK silencing alleles and their critical SNVs by single‐molecule sequencing and the findings were confirmed by transfection and expression studies.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

et al. 2023

View full text Add to dashboard Cite

show abstract

“…Novel techniques were applied to RBC antigen genotyping to decrease error rates and cost. Zhang et al utilized long‐read NGS, as opposed to typical short reads, combined with a custom bioinformatics pipeline, to fully assemble the RH region 37 …”

Section: Immunohematology and Genomicsmentioning

confidence: 99%

“…Zhang et al utilized long-read NGS, as opposed to typical short reads, combined with a custom bioinformatics pipeline, to fully assemble the RH region. 37 Nanopore-based sequencing technology can be less expensive than other NGS methods and allows for portability. Tounsi et al used this method to perform RHD genotyping on 13 samples.…”

Section: Genomic Sequencingmentioning

confidence: 99%

Current advances in 2022: A critical review of selected topics by the Association for the Advancement of Blood and Biotherapies (AABB) Clinical Transfusion Medicine Committee

et al. 2023

View full text Add to dashboard Cite

BackgroundThe Association for the Advancement of Blood and Biotherapies Clinical Transfusion Medicine Committee (CTMC) composes a summary of new and important advances in transfusion medicine (TM) on an annual basis. Since 2018, this has been assembled into a manuscript and published in Transfusion.Study design and methodsCTMC members selected original manuscripts relevant to TM that were published electronically and/or in print during calendar year 2022. Papers were selected based on perceived importance and/or originality. References for selected papers were made available to CTMC members to provide feedback. Members were also encouraged to identify papers that may have been omitted initially. They then worked in groups of two to three to write a summary for each new publication within their broader topic. Each topic summary was then reviewed and edited by two separate committee members. The final manuscript was assembled by the first and senior authors. While this review is extensive, it is not a systematic review and some publications considered important by readers may have been excluded.ResultsFor calendar year 2022, summaries of key publications were assembled for the following broader topics within TM: blood component therapy; infectious diseases, blood donor testing, and collections; patient blood management; immunohematology and genomics; hemostasis; hemoglobinopathies; apheresis and cell therapy; pediatrics; and health care disparities, diversity, equity, and inclusion.DiscussionThis Committee Report reviews and summarizes important publications and advances in TM published during calendar year 2022, and maybe a useful educational tool.

show abstract

“…Chimeras (Figure 1) are considered as the results of mis-priming during the MDA 33, 36 , appear as DNA rearrangements, cannot be used for genome assembly 34, 37–40 , and make confusion in genetic variation analysis 41–44 . The present of chimeras is an unevadable problem of MDA and has caught more and more attentions 45 46–48 .…”

Section: Introductionmentioning

confidence: 99%

3^rd-ChimeraMiner: A pipeline for integrated analysis of whole genome amplification generated chimeric sequences using long-read sequencing

Lü¹,

Qiao²,

An³

et al. 2022

Preprint

View full text Add to dashboard Cite

Multiple displacement amplification (MDA) has become one of the most commonly used method of whole genome amplification (WGA) due to the high processivity, strand displacement capacity and high fidelity of the phi29 DNA polymerase, MDA generate vast amount of DNA with higher molecules weight (up to 100kb) and greater genome coverage. Along with the development of the sequencing platform, it is possible to sequence the MDA-amplified DNA molecules with over 20kb by long-read sequencing. However, one of the challenges is the formation of chimeras, which exist in all MDA products, and seriously interfere with the downstream analysis of the long-read sequencing data of MDA-amplified DNA. In this study, we constructed 3rd-ChimeraMiner, a chimera detection pipeline for analyzing the long-read sequencing of MDA products, recognizing chimeras, and integrating chimeras into the downstream analysis. Five sequencing data of MDA with different magnification fold were analyzed in here, the proportions of chimeras are much higher than that of next-generation sequencing reads and increase with the increase of magnification folds, ranging from 42% to over 76%. After comparing, 99.92% of recognized chimeras have been demonstrated not to exist in original genomes. After detecting chimeras by 3rd-ChimeraMiner, the full-length mapping ratio increased, means more PacBio data could be used in downstream analysis, and mean 97.77% inversions were removed after transferred chimeras into normal reads. 3rd-ChimeraMiner revealed efficiency and accuracy in discovering chimeras from long-read sequencing data of MDA, and is promising to be widely used in single-cell sequencing.

show abstract

Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion

Cited by 20 publications

References 49 publications

Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

Current advances in 2022: A critical review of selected topics by the Association for the Advancement of Blood and Biotherapies (AABB) Clinical Transfusion Medicine Committee

3^rd-ChimeraMiner: A pipeline for integrated analysis of whole genome amplification generated chimeric sequences using long-read sequencing

Contact Info

Product

Resources

About

Accurate long-read sequencing allows assembly of the duplicated RHD and RHCE genes harboring variants relevant to blood transfusion

Cited by 20 publications

References 49 publications

Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

Current advances in 2022: A critical review of selected topics by the Association for the Advancement of Blood and Biotherapies (AABB) Clinical Transfusion Medicine Committee

3rd-ChimeraMiner: A pipeline for integrated analysis of whole genome amplification generated chimeric sequences using long-read sequencing

Contact Info

Product

Resources

About

3^rd-ChimeraMiner: A pipeline for integrated analysis of whole genome amplification generated chimeric sequences using long-read sequencing