Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing
A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with l...
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creator | Lange, Vinzenz Böhme, Irina Hofmann, Jan Lang, Kathrin Sauter, Jürgen Schöne, Bianca Paul, Patrick Albrecht, Viviane Andreas, Johanna M Baier, Daniel M Nething, Jochen Ehninger, Ulf Schwarzelt, Carmen Pingel, Julia Ehninger, Gerhard Schmidt, Alexander H |
description | A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing.
We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week.
The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration. |
doi_str_mv | 10.1186/1471-2164-15-63 |
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We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week.
The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.</description><identifier>ISSN: 1471-2164</identifier><identifier>EISSN: 1471-2164</identifier><identifier>DOI: 10.1186/1471-2164-15-63</identifier><identifier>PMID: 24460756</identifier><language>eng</language><publisher>England: BioMed Central Ltd</publisher><subject>Alleles ; Analysis ; Data analysis ; Data processing ; Deoxyribonucleic acid ; DNA ; DNA - analysis ; DNA - isolation & purification ; DNA Primers - metabolism ; DNA sequencing ; Economic aspects ; Equipment and supplies ; Exons ; Genes ; Genomics ; Histocompatibility Testing - economics ; Histocompatibility Testing - instrumentation ; HLA Antigens - genetics ; Humans ; Methodology ; Methods ; Microfluidic Analytical Techniques ; Nucleotide sequencing ; Polymerase Chain Reaction ; Pumping machinery industry ; Sequence Analysis, DNA ; Stem cells</subject><ispartof>BMC genomics, 2014-01, Vol.15 (1), p.63-63, Article 63</ispartof><rights>COPYRIGHT 2014 BioMed Central Ltd.</rights><rights>2014 Lange et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.</rights><rights>Copyright © 2014 Lange et al.; licensee BioMed Central Ltd. 2014 Lange et al.; licensee BioMed Central Ltd.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c621t-791cfe044a6e236a0bce779f78b67b432bfefc7ad320b384057d42f832ba2243</citedby><cites>FETCH-LOGICAL-c621t-791cfe044a6e236a0bce779f78b67b432bfefc7ad320b384057d42f832ba2243</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909933/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC3909933/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,864,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/24460756$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Lange, Vinzenz</creatorcontrib><creatorcontrib>Böhme, Irina</creatorcontrib><creatorcontrib>Hofmann, Jan</creatorcontrib><creatorcontrib>Lang, Kathrin</creatorcontrib><creatorcontrib>Sauter, Jürgen</creatorcontrib><creatorcontrib>Schöne, Bianca</creatorcontrib><creatorcontrib>Paul, Patrick</creatorcontrib><creatorcontrib>Albrecht, Viviane</creatorcontrib><creatorcontrib>Andreas, Johanna M</creatorcontrib><creatorcontrib>Baier, Daniel M</creatorcontrib><creatorcontrib>Nething, Jochen</creatorcontrib><creatorcontrib>Ehninger, Ulf</creatorcontrib><creatorcontrib>Schwarzelt, Carmen</creatorcontrib><creatorcontrib>Pingel, Julia</creatorcontrib><creatorcontrib>Ehninger, Gerhard</creatorcontrib><creatorcontrib>Schmidt, Alexander H</creatorcontrib><title>Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing</title><title>BMC genomics</title><addtitle>BMC Genomics</addtitle><description>A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing.
We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week.
The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.</description><subject>Alleles</subject><subject>Analysis</subject><subject>Data analysis</subject><subject>Data processing</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA - analysis</subject><subject>DNA - isolation & purification</subject><subject>DNA Primers - metabolism</subject><subject>DNA sequencing</subject><subject>Economic aspects</subject><subject>Equipment and supplies</subject><subject>Exons</subject><subject>Genes</subject><subject>Genomics</subject><subject>Histocompatibility Testing - economics</subject><subject>Histocompatibility Testing - instrumentation</subject><subject>HLA Antigens - genetics</subject><subject>Humans</subject><subject>Methodology</subject><subject>Methods</subject><subject>Microfluidic Analytical Techniques</subject><subject>Nucleotide sequencing</subject><subject>Polymerase Chain Reaction</subject><subject>Pumping machinery industry</subject><subject>Sequence Analysis, DNA</subject><subject>Stem cells</subject><issn>1471-2164</issn><issn>1471-2164</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><recordid>eNqFks1r3DAQxU1pSdI0596KoZf04ETfsi-FZWmawJZCk7uQtSNbwbYcSy7d_z4ySbfZUig6jJj5zWN4vCx7j9EFxqW4xEzigmDBCswLQV9lJ_vO6xf_4-xtCPcIYVkSfpQdE8YEklycZFdrH2IB1jrjYIh565q2iO3k56Yd55hfb1Z53I1uaPJ6l39zt_CQ637snPFDHuBhhsGk4bvsjdVdgLPneprdXX25W18Xm-9fb9arTWEEwbGQFTYWEGNaAKFCo9qAlJWVZS1kzSipLVgj9ZYSVNOSIS63jNgyDTQhjJ5mn59kx7nuYWvSxZPu1Di5Xk875bVTh5PBtarxPxWtUFVRmgTOnwUmn24PUfUuGOg6PYCfg8IcoeSRlOL_KKsqjLnAMqEf_0Lv_TwNyYiFYpgSyfEfqtEdKDdYn040i6hacVpxwUu2aF38g0pvC_1iOliX-gcLnw4WEhPhV2z0HIK6uf1xyF4-sWbyIUxg99ZhpJZAqSUyaolMskKJxbAPLx3f878TRB8Bcg_CrA</recordid><startdate>20140124</startdate><enddate>20140124</enddate><creator>Lange, Vinzenz</creator><creator>Böhme, Irina</creator><creator>Hofmann, Jan</creator><creator>Lang, Kathrin</creator><creator>Sauter, Jürgen</creator><creator>Schöne, Bianca</creator><creator>Paul, Patrick</creator><creator>Albrecht, Viviane</creator><creator>Andreas, Johanna M</creator><creator>Baier, Daniel M</creator><creator>Nething, Jochen</creator><creator>Ehninger, Ulf</creator><creator>Schwarzelt, Carmen</creator><creator>Pingel, Julia</creator><creator>Ehninger, Gerhard</creator><creator>Schmidt, Alexander H</creator><general>BioMed Central Ltd</general><general>BioMed Central</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>ISR</scope><scope>3V.</scope><scope>7QP</scope><scope>7QR</scope><scope>7SS</scope><scope>7TK</scope><scope>7U7</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M7P</scope><scope>P64</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20140124</creationdate><title>Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing</title><author>Lange, Vinzenz ; Böhme, Irina ; Hofmann, Jan ; Lang, Kathrin ; Sauter, Jürgen ; Schöne, Bianca ; Paul, Patrick ; Albrecht, Viviane ; Andreas, Johanna M ; Baier, Daniel M ; Nething, Jochen ; Ehninger, Ulf ; Schwarzelt, Carmen ; Pingel, Julia ; Ehninger, Gerhard ; Schmidt, Alexander H</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c621t-791cfe044a6e236a0bce779f78b67b432bfefc7ad320b384057d42f832ba2243</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Alleles</topic><topic>Analysis</topic><topic>Data analysis</topic><topic>Data processing</topic><topic>Deoxyribonucleic acid</topic><topic>DNA</topic><topic>DNA - analysis</topic><topic>DNA - isolation & purification</topic><topic>DNA Primers - metabolism</topic><topic>DNA sequencing</topic><topic>Economic aspects</topic><topic>Equipment and supplies</topic><topic>Exons</topic><topic>Genes</topic><topic>Genomics</topic><topic>Histocompatibility Testing - economics</topic><topic>Histocompatibility Testing - instrumentation</topic><topic>HLA Antigens - genetics</topic><topic>Humans</topic><topic>Methodology</topic><topic>Methods</topic><topic>Microfluidic Analytical Techniques</topic><topic>Nucleotide sequencing</topic><topic>Polymerase Chain Reaction</topic><topic>Pumping machinery industry</topic><topic>Sequence Analysis, DNA</topic><topic>Stem cells</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Lange, Vinzenz</creatorcontrib><creatorcontrib>Böhme, Irina</creatorcontrib><creatorcontrib>Hofmann, Jan</creatorcontrib><creatorcontrib>Lang, Kathrin</creatorcontrib><creatorcontrib>Sauter, Jürgen</creatorcontrib><creatorcontrib>Schöne, Bianca</creatorcontrib><creatorcontrib>Paul, Patrick</creatorcontrib><creatorcontrib>Albrecht, Viviane</creatorcontrib><creatorcontrib>Andreas, Johanna M</creatorcontrib><creatorcontrib>Baier, Daniel M</creatorcontrib><creatorcontrib>Nething, Jochen</creatorcontrib><creatorcontrib>Ehninger, Ulf</creatorcontrib><creatorcontrib>Schwarzelt, Carmen</creatorcontrib><creatorcontrib>Pingel, Julia</creatorcontrib><creatorcontrib>Ehninger, Gerhard</creatorcontrib><creatorcontrib>Schmidt, Alexander H</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Gale In Context: Science</collection><collection>ProQuest Central (Corporate)</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Neurosciences Abstracts</collection><collection>Toxicology Abstracts</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>ProQuest Pharma Collection</collection><collection>Technology Research Database</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>Environmental Sciences and Pollution Management</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Engineering Research Database</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Biological Science Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Access via ProQuest (Open Access)</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>BMC genomics</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Lange, Vinzenz</au><au>Böhme, Irina</au><au>Hofmann, Jan</au><au>Lang, Kathrin</au><au>Sauter, Jürgen</au><au>Schöne, Bianca</au><au>Paul, Patrick</au><au>Albrecht, Viviane</au><au>Andreas, Johanna M</au><au>Baier, Daniel M</au><au>Nething, Jochen</au><au>Ehninger, Ulf</au><au>Schwarzelt, Carmen</au><au>Pingel, Julia</au><au>Ehninger, Gerhard</au><au>Schmidt, Alexander H</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing</atitle><jtitle>BMC genomics</jtitle><addtitle>BMC Genomics</addtitle><date>2014-01-24</date><risdate>2014</risdate><volume>15</volume><issue>1</issue><spage>63</spage><epage>63</epage><pages>63-63</pages><artnum>63</artnum><issn>1471-2164</issn><eissn>1471-2164</eissn><abstract>A close match of the HLA alleles between donor and recipient is an important prerequisite for successful unrelated hematopoietic stem cell transplantation. To increase the chances of finding an unrelated donor, registries recruit many hundred thousands of volunteers each year. Many registries with limited resources have had to find a trade-off between cost and resolution and extent of typing for newly recruited donors in the past. Therefore, we have taken advantage of recent improvements in NGS to develop a workflow for low-cost, high-resolution HLA typing.
We have established a straightforward three-step workflow for high-throughput HLA typing: Exons 2 and 3 of HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 are amplified by PCR on Fluidigm Access Array microfluidic chips. Illumina sequencing adapters and sample specific tags are directly incorporated during PCR. Upon pooling and cleanup, 384 samples are sequenced in a single Illumina MiSeq run. We developed "neXtype" for streamlined data analysis and HLA allele assignment. The workflow was validated with 1140 samples typed at 6 loci. All neXtype results were concordant with the Sanger sequences, demonstrating error-free typing of more than 6000 HLA loci. Current capacity in routine operation is 12,000 samples per week.
The workflow presented proved to be a cost-efficient alternative to Sanger sequencing for high-throughput HLA typing. Despite the focus on cost efficiency, resolution exceeds the current standards of Sanger typing for donor registration.</abstract><cop>England</cop><pub>BioMed Central Ltd</pub><pmid>24460756</pmid><doi>10.1186/1471-2164-15-63</doi><tpages>1</tpages><oa>free_for_read</oa></addata></record> |
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subjects | Alleles Analysis Data analysis Data processing Deoxyribonucleic acid DNA DNA - analysis DNA - isolation & purification DNA Primers - metabolism DNA sequencing Economic aspects Equipment and supplies Exons Genes Genomics Histocompatibility Testing - economics Histocompatibility Testing - instrumentation HLA Antigens - genetics Humans Methodology Methods Microfluidic Analytical Techniques Nucleotide sequencing Polymerase Chain Reaction Pumping machinery industry Sequence Analysis, DNA Stem cells |
title | Cost-efficient high-throughput HLA typing by MiSeq amplicon sequencing |
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