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

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 - PLoS ONE (2016)

Bottom Line: Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity--the concordance rate.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.

View Article: PubMed Central - PubMed

Affiliation: Department for Congenital Disorders, Danish Centre for Neonatal Screening, Section of Neonatal Genetics, Statens Serum Institut, Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus

Study design.This study aims to identify systematic differences between DBS and WB by testing both sample types from a number of individuals. Pilot 1, Pilot 2 and Pilot 3 included seven, eight and seven subjects represented with a neonatal DBS sample and corresponding adult WB reference sample each, respectively. A minimum of two sample types per subject were prepared for library preparation: wgaDNA of DNA from 2x3.2 mm of neonatal DBSs (DBS_2×3.2) and raw control DNA from the WB reference sample (WB_ref). Pilot 2 also included a replicate sample of each of the WB_ref samples (WB_ref_replicate)(replicate sample not shown in the cartoon). Pilot 3 included two additional sample types; WGA of DNA from 2x1.6 mm neonatal DBS (DBS_2x1.6) and WGA of the WB reference sample (WB_WGA_ref). Note that the DBS_2x1.6 sample type of Pilot 3 was prepared and sequenced in triplicate using different sets of 2x1.6 mm discs for all of the subjects included. The samples were set up for library preparation using different kits in the respective pilot studies, before sequencing and data analysis. Please note the color-coding used with green, blue and orange specifying the respective pilot studies. All samples were retrieved from the Danish Neonatal Screening Biobank.
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pone.0153253.g001: Study design.This study aims to identify systematic differences between DBS and WB by testing both sample types from a number of individuals. Pilot 1, Pilot 2 and Pilot 3 included seven, eight and seven subjects represented with a neonatal DBS sample and corresponding adult WB reference sample each, respectively. A minimum of two sample types per subject were prepared for library preparation: wgaDNA of DNA from 2x3.2 mm of neonatal DBSs (DBS_2×3.2) and raw control DNA from the WB reference sample (WB_ref). Pilot 2 also included a replicate sample of each of the WB_ref samples (WB_ref_replicate)(replicate sample not shown in the cartoon). Pilot 3 included two additional sample types; WGA of DNA from 2x1.6 mm neonatal DBS (DBS_2x1.6) and WGA of the WB reference sample (WB_WGA_ref). Note that the DBS_2x1.6 sample type of Pilot 3 was prepared and sequenced in triplicate using different sets of 2x1.6 mm discs for all of the subjects included. The samples were set up for library preparation using different kits in the respective pilot studies, before sequencing and data analysis. Please note the color-coding used with green, blue and orange specifying the respective pilot studies. All samples were retrieved from the Danish Neonatal Screening Biobank.

Mentions: Fig 1 provides a detailed overview of the study design and sample preparation used in the respective pilots. Note that all details of sample preparation relevant to differentiate between DNA sample types are described herein. The sample types prepared were for Pilot 1: DBS_2×3.2 and WB_ref sample types; Pilot 2: DBS_2×3.2, WB_ref and WB_ref_replica sample types and Pilot 3: DBS_2×1.6, DBS_2×3.2, WB_WGA_ref and corresponding WB_ ref sample types. DNA extractions of the WB reference samples were with the Maxwell 16 LEV Blood DNA Kit (Promega) as prescribed by the manufacturer, and DNA extractions of the neonatal DBS samples were with the Extract-N-Amp Blood PCR Kit (Sigma) as described previously [1]. WGA was carried out in triplicate using the REPLIg Kit (QIAGEN) as has also been described previously [1]. The DNA was quantified using the Quant-IT PicoGreen dsDNA Reagent (Invitrogen) before library preparation and sequencing.


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

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 - PLoS ONE (2016)

Study design.This study aims to identify systematic differences between DBS and WB by testing both sample types from a number of individuals. Pilot 1, Pilot 2 and Pilot 3 included seven, eight and seven subjects represented with a neonatal DBS sample and corresponding adult WB reference sample each, respectively. A minimum of two sample types per subject were prepared for library preparation: wgaDNA of DNA from 2x3.2 mm of neonatal DBSs (DBS_2×3.2) and raw control DNA from the WB reference sample (WB_ref). Pilot 2 also included a replicate sample of each of the WB_ref samples (WB_ref_replicate)(replicate sample not shown in the cartoon). Pilot 3 included two additional sample types; WGA of DNA from 2x1.6 mm neonatal DBS (DBS_2x1.6) and WGA of the WB reference sample (WB_WGA_ref). Note that the DBS_2x1.6 sample type of Pilot 3 was prepared and sequenced in triplicate using different sets of 2x1.6 mm discs for all of the subjects included. The samples were set up for library preparation using different kits in the respective pilot studies, before sequencing and data analysis. Please note the color-coding used with green, blue and orange specifying the respective pilot studies. All samples were retrieved from the Danish Neonatal Screening Biobank.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835089&req=5

pone.0153253.g001: Study design.This study aims to identify systematic differences between DBS and WB by testing both sample types from a number of individuals. Pilot 1, Pilot 2 and Pilot 3 included seven, eight and seven subjects represented with a neonatal DBS sample and corresponding adult WB reference sample each, respectively. A minimum of two sample types per subject were prepared for library preparation: wgaDNA of DNA from 2x3.2 mm of neonatal DBSs (DBS_2×3.2) and raw control DNA from the WB reference sample (WB_ref). Pilot 2 also included a replicate sample of each of the WB_ref samples (WB_ref_replicate)(replicate sample not shown in the cartoon). Pilot 3 included two additional sample types; WGA of DNA from 2x1.6 mm neonatal DBS (DBS_2x1.6) and WGA of the WB reference sample (WB_WGA_ref). Note that the DBS_2x1.6 sample type of Pilot 3 was prepared and sequenced in triplicate using different sets of 2x1.6 mm discs for all of the subjects included. The samples were set up for library preparation using different kits in the respective pilot studies, before sequencing and data analysis. Please note the color-coding used with green, blue and orange specifying the respective pilot studies. All samples were retrieved from the Danish Neonatal Screening Biobank.
Mentions: Fig 1 provides a detailed overview of the study design and sample preparation used in the respective pilots. Note that all details of sample preparation relevant to differentiate between DNA sample types are described herein. The sample types prepared were for Pilot 1: DBS_2×3.2 and WB_ref sample types; Pilot 2: DBS_2×3.2, WB_ref and WB_ref_replica sample types and Pilot 3: DBS_2×1.6, DBS_2×3.2, WB_WGA_ref and corresponding WB_ ref sample types. DNA extractions of the WB reference samples were with the Maxwell 16 LEV Blood DNA Kit (Promega) as prescribed by the manufacturer, and DNA extractions of the neonatal DBS samples were with the Extract-N-Amp Blood PCR Kit (Sigma) as described previously [1]. WGA was carried out in triplicate using the REPLIg Kit (QIAGEN) as has also been described previously [1]. The DNA was quantified using the Quant-IT PicoGreen dsDNA Reagent (Invitrogen) before library preparation and sequencing.

Bottom Line: Following sequencing and data analysis, we compared pairwise variant calls to obtain a measure of similarity--the concordance rate.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.

View Article: PubMed Central - PubMed

Affiliation: Department for Congenital Disorders, Danish Centre for Neonatal Screening, Section of Neonatal Genetics, Statens Serum Institut, Copenhagen, Denmark.

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.

No MeSH data available.


Related in: MedlinePlus