High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

Jesper Buchhave Poulsen; Francesco Lescai; Jakob Grove; Marie Bækvad-Hansen; Michael Christiansen; Christian Munch Hagen; Julian Maller; Christine Stevens; Shenting Li; Qibin Li; Jihua Sun; Jun Wang; Merete Nordentoft; Thomas Mears Werge; Preben Bo Mortensen; Anders Dupont Børglum; Mark Daly; David Michael Hougaard; Jonas Bybjerg-Grauholm; Mads Vilhelm Hollegaard

doi:10.1371/journal.pone.0153253

High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

Jesper Buchhave Poulsen, Francesco Lescai, Jakob Grove, Marie Bækvad-Hansen, Michael Christiansen, Christian Munch Hagen, Julian Maller, Christine Stevens, Shenting Li, Qibin Li, Jihua Sun, Jun Wang, Merete Nordentoft, Thomas Mears Werge, Preben Bo Mortensen, Anders Dupont Børglum, Mark Daly, David Michael Hougaard, Jonas Bybjerg-Grauholm, Mads Vilhelm Hollegaard

Department of Clinical Medicine

20 Citations (Scopus)

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Abstract

Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity--the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference--whole-blood DNA--based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.

Original language	English
Article number	e0153253
Journal	P L o S One
Volume	11
Issue number	4
Number of pages	13
ISSN	1932-6203
DOIs	https://doi.org/10.1371/journal.pone.0153253
Publication status	Published - Apr 2016

Keywords

Adult
Dried Blood Spot Testing
Exome
Genome, Human
High-Throughput Nucleotide Sequencing
Humans
Infant, Newborn
Nucleic Acid Amplification Techniques
Pilot Projects
Polymorphism, Single Nucleotide
Reproducibility of Results

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10.1371/journal.pone.0153253Licence: CC BY

High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNAFinal published version, 1.04 MBLicence: CC BY

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Poulsen, J. B., Lescai, F., Grove, J., Bækvad-Hansen, M., Christiansen, M., Hagen, C. M., Maller, J., Stevens, C., Li, S., Li, Q., Sun, J., Wang, J., Nordentoft, M., Werge, T. M., Mortensen, P. B., Børglum, A. D., Daly, M., Hougaard, D. M., Bybjerg-Grauholm, J., & Hollegaard, M. V. (2016). High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA. P L o S One, 11(4), [e0153253]. https://doi.org/10.1371/journal.pone.0153253

Poulsen, JB, Lescai, F, Grove, J, Bækvad-Hansen, M, Christiansen, M, Hagen, CM, Maller, J, Stevens, C, Li, S, Li, Q, Sun, J, Wang, J, Nordentoft, M , Werge, TM, Mortensen, PB, Børglum, AD, Daly, M, Hougaard, DM, Bybjerg-Grauholm, J & Hollegaard, MV 2016, 'High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA', P L o S One, vol. 11, no. 4, e0153253. https://doi.org/10.1371/journal.pone.0153253

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title = "High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA",

abstract = "Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity--the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference--whole-blood DNA--based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.",

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AU - Poulsen, Jesper Buchhave

AU - Lescai, Francesco

AU - Grove, Jakob

AU - Bækvad-Hansen, Marie

AU - Christiansen, Michael

AU - Hagen, Christian Munch

AU - Maller, Julian

AU - Stevens, Christine

AU - Li, Shenting

AU - Li, Qibin

AU - Sun, Jihua

AU - Wang, Jun

AU - Nordentoft, Merete

AU - Werge, Thomas Mears

AU - Mortensen, Preben Bo

AU - Børglum, Anders Dupont

AU - Daly, Mark

AU - Hougaard, David Michael

AU - Bybjerg-Grauholm, Jonas

AU - Hollegaard, Mads Vilhelm

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N2 - Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity--the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference--whole-blood DNA--based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.

AB - Stored neonatal dried blood spot (DBS) samples from neonatal screening programmes are a valuable diagnostic and research resource. Combined with information from national health registries they can be used in population-based studies of genetic diseases. DNA extracted from neonatal DBSs can be amplified to obtain micrograms of an otherwise limited resource, referred to as whole-genome amplified DNA (wgaDNA). Here we investigate the robustness of exome sequencing of wgaDNA of neonatal DBS samples. We conducted three pilot studies of seven, eight and seven subjects, respectively. For each subject we analysed a neonatal DBS sample and corresponding adult whole-blood (WB) reference sample. Different DNA sample types were prepared for each of the subjects. Pilot 1: wgaDNA of 2x3.2mm neonatal DBSs (DBS_2x3.2) and raw DNA extract of the WB reference sample (WB_ref). Pilot 2: DBS_2x3.2, WB_ref and a WB_ref replica sharing DNA extract with the WB_ref sample. Pilot 3: DBS_2x3.2, WB_ref, wgaDNA of 2x1.6 mm neonatal DBSs and wgaDNA of the WB reference sample. Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity--the concordance rate. Concordance rates were slightly lower when comparing DBS vs WB sample types than for any two WB sample types of the same subject before filtering of the variant calls. The overall concordance rates were dependent on the variant type, with SNPs performing best. Post-filtering, the comparisons of DBS vs WB and WB vs WB sample types yielded similar concordance rates, with values close to 100%. WgaDNA of neonatal DBS samples performs with great accuracy and efficiency in exome sequencing. The wgaDNA performed similarly to matched high-quality reference--whole-blood DNA--based on concordance rates calculated from variant calls. No differences were observed substituting 2x3.2 with 2x1.6 mm discs, allowing for additional reduction of sample material in future projects.

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KW - High-Throughput Nucleotide Sequencing

KW - Humans

KW - Infant, Newborn

KW - Nucleic Acid Amplification Techniques

KW - Pilot Projects

KW - Polymorphism, Single Nucleotide

KW - Reproducibility of Results

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DO - 10.1371/journal.pone.0153253

M3 - Journal article

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SN - 1932-6203

VL - 11

JO - PLoS Computational Biology

JF - PLoS Computational Biology

IS - 4

M1 - e0153253

ER -

High-Quality Exome Sequencing of Whole-Genome Amplified Neonatal Dried Blood Spot DNA

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Keywords

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