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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

Comparison of sample types from variant calls—concordance rates.The concordance rates were calculated by pairwise comparison of variant calls before (upper panels) and after filtering (lower panels). The sample types compared were DBS_2x3.2 vs WB_ref in Pilot 1 (A and B), DBS_2x3.2 vs WB_ref and WB_ref vs WB_ref replica in Pilot 2 (C and D) and DBS_2x1.6 vs WB_ref, DBS_2x3.2 vs WB_ref, DBS_2x3.2 vs DBS_2x1.6 and WB_ref vs WB_WGA_ref in Pilot 3 (E and F). The rates have been presented per variant type: SNP, insertion, deletion and multiallelic calls, and comprise the averages of all comparisons made for a given sample pair, corresponding to the number of subjects in the pilot, i.e. Pilot 1 = 7, Pilot 2 = 8 and Pilot 3 = 7. Note that for comparisons using the DBS_2x1.6 sample type (see the fig), each individual replica was firstly compared to the WB_ref or DBS_2x3.2 sample types followed by the calculation of average values hereof, which were used in the figure.
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pone.0153253.g003: Comparison of sample types from variant calls—concordance rates.The concordance rates were calculated by pairwise comparison of variant calls before (upper panels) and after filtering (lower panels). The sample types compared were DBS_2x3.2 vs WB_ref in Pilot 1 (A and B), DBS_2x3.2 vs WB_ref and WB_ref vs WB_ref replica in Pilot 2 (C and D) and DBS_2x1.6 vs WB_ref, DBS_2x3.2 vs WB_ref, DBS_2x3.2 vs DBS_2x1.6 and WB_ref vs WB_WGA_ref in Pilot 3 (E and F). The rates have been presented per variant type: SNP, insertion, deletion and multiallelic calls, and comprise the averages of all comparisons made for a given sample pair, corresponding to the number of subjects in the pilot, i.e. Pilot 1 = 7, Pilot 2 = 8 and Pilot 3 = 7. Note that for comparisons using the DBS_2x1.6 sample type (see the fig), each individual replica was firstly compared to the WB_ref or DBS_2x3.2 sample types followed by the calculation of average values hereof, which were used in the figure.

Mentions: To evaluate sample performance we executed a pairwise comparison of genotypes based on the variant calls resulting in a similarity measure between different sample types—the concordance rate (Fig 3). Concordance rates were obtained both before and after filtering of the calls and grouped by variant type into SNPs, insertions, deletions and multiallelic calls. The numbers supporting the graphs in Fig 3 are available in table format in S2 Table. We compared the DBS_2x3.2 and WB_ref sample types (all pilots) to obtain a measure of the feasibility of sequencing neonatal DBS samples. The WB_ref and WB_ref replica sample types of Pilot 2 were compared to establish a high-quality standard used for comparison to the DBS_2x3.2 vs WB_ref comparison, thereby testing the relative performance of the DBS_2x3.2 sample type. The DBS_2x1.6 vs WB_ref and DBS_2x3.2 vs DBS_2x1.6 comparisons of Pilot 3 were made in order to evaluate the potential of reducing the disc size, and the WB_ref vs WB_WGA_ref comparison for probing an effect resulting from the amplification reaction. A comparison of the DBS_2x1.6 sample type replicas with the WB_ref sample type, i.e. replica_1 vs WB_ref, replica_2 vs WB_ref and replica_3 vs WB_ref yielded similar concordance rates (see S1 Fig). Based on this, we decided to include all of the data of the DBS_2x1.6 sample type for calculation of the concordances displayed in Fig 3; that is the DBS_2x1.6 vs WB_ref and DBS_2x3.2 vs DBS_2x1.6 comparisons, respectively. This was done by initial comparison of each of the DBS_2x1.6 sample type replicas with the WB_ref or DBS_2x3.2 sample types, followed by the calculation of average values hereof. An initial look at the concordances in Fig 3 before filtering show strong dependency by variant type with SNPs > insertions > deletions > mulitiallelic calls, respectively (upper panels). This is similar to what has previously been observed in WES studies splitting calls by variant type, and results due to well-known difficulties of calling high-complexity variants as well as a general higher probability of encountering sequence variation between any two samples for variants of increasing lengths [26]. The pre-filtered concordance rates are consistent with other exome sequencing experiments dealing with sample replicas: previous reported rates were in the range of 82–92%, the numbers being largely dependent on the depth of coverage, as is also reported here [27]. Notably, we observe somewhat lower concordance rates of the DBS vs WB comparisons (i.e. all comparisons of neonatal DBS samples and WB reference samples) over the WB vs WB comparisons (all comparisons of WB reference samples) before filtering in Pilot 2 and Pilot 3, which may be ascribed to subtle differences in overall DNA quality of DBS-derived wgaDNA as compared to WB-derived DNA. This difference become neutralized after filtering yielding similar concordance rates between the DBS vs WB and WB vs WB comparisons independent of whether 2x3.2 mm or 2x1.6 mm discs were used for DNA extraction. Moreover, post-filtering the concordance rates of all pairwise compared samples are very close to 100% (the multiallelic calls of Pilots 1 and 3 omitted, for which rates only reaches 85–88%). This shows that DBS-derived wgaDNA no matter the input quantities, i.e. 2x3.2 mm or 2x1.6 mm discs can be set to perform similarly to high-quality DNA from a WB reference sample, if in case the proper filter settings are applied. This validates the use of DNA of neonatal DBSs for WES. The one most striking example illustrating the feasibility of sequencing the neonatal DNA can be inferred from the WB_ref vs WB_ref replica and DBS_2x3.2 vs WB_ref comparisons of Pilot 2. The fact that duplicate sequencing data produced using the same high-quality DNA (e.g. the WB_ref vs WB_ref replica comparison), scores similarly to that DNA set against wgaDNA of a neonatal DBS sample, proves the validity of the latter. One concern of using wgaDNA of neonatal DBS samples has been if the amplification reaction would introduce sequence errors and allele dropouts caused by the low inputs and presumed low quality of the DNA used. However, based on the very high similarities observed between the DBS vs WB and WB vs WB comparisons along with the very high concordances overall post-filtering, we conclude that this does not seem to constitute a problem.


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)

Comparison of sample types from variant calls—concordance rates.The concordance rates were calculated by pairwise comparison of variant calls before (upper panels) and after filtering (lower panels). The sample types compared were DBS_2x3.2 vs WB_ref in Pilot 1 (A and B), DBS_2x3.2 vs WB_ref and WB_ref vs WB_ref replica in Pilot 2 (C and D) and DBS_2x1.6 vs WB_ref, DBS_2x3.2 vs WB_ref, DBS_2x3.2 vs DBS_2x1.6 and WB_ref vs WB_WGA_ref in Pilot 3 (E and F). The rates have been presented per variant type: SNP, insertion, deletion and multiallelic calls, and comprise the averages of all comparisons made for a given sample pair, corresponding to the number of subjects in the pilot, i.e. Pilot 1 = 7, Pilot 2 = 8 and Pilot 3 = 7. Note that for comparisons using the DBS_2x1.6 sample type (see the fig), each individual replica was firstly compared to the WB_ref or DBS_2x3.2 sample types followed by the calculation of average values hereof, which were used in the figure.
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Related In: Results  -  Collection

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

pone.0153253.g003: Comparison of sample types from variant calls—concordance rates.The concordance rates were calculated by pairwise comparison of variant calls before (upper panels) and after filtering (lower panels). The sample types compared were DBS_2x3.2 vs WB_ref in Pilot 1 (A and B), DBS_2x3.2 vs WB_ref and WB_ref vs WB_ref replica in Pilot 2 (C and D) and DBS_2x1.6 vs WB_ref, DBS_2x3.2 vs WB_ref, DBS_2x3.2 vs DBS_2x1.6 and WB_ref vs WB_WGA_ref in Pilot 3 (E and F). The rates have been presented per variant type: SNP, insertion, deletion and multiallelic calls, and comprise the averages of all comparisons made for a given sample pair, corresponding to the number of subjects in the pilot, i.e. Pilot 1 = 7, Pilot 2 = 8 and Pilot 3 = 7. Note that for comparisons using the DBS_2x1.6 sample type (see the fig), each individual replica was firstly compared to the WB_ref or DBS_2x3.2 sample types followed by the calculation of average values hereof, which were used in the figure.
Mentions: To evaluate sample performance we executed a pairwise comparison of genotypes based on the variant calls resulting in a similarity measure between different sample types—the concordance rate (Fig 3). Concordance rates were obtained both before and after filtering of the calls and grouped by variant type into SNPs, insertions, deletions and multiallelic calls. The numbers supporting the graphs in Fig 3 are available in table format in S2 Table. We compared the DBS_2x3.2 and WB_ref sample types (all pilots) to obtain a measure of the feasibility of sequencing neonatal DBS samples. The WB_ref and WB_ref replica sample types of Pilot 2 were compared to establish a high-quality standard used for comparison to the DBS_2x3.2 vs WB_ref comparison, thereby testing the relative performance of the DBS_2x3.2 sample type. The DBS_2x1.6 vs WB_ref and DBS_2x3.2 vs DBS_2x1.6 comparisons of Pilot 3 were made in order to evaluate the potential of reducing the disc size, and the WB_ref vs WB_WGA_ref comparison for probing an effect resulting from the amplification reaction. A comparison of the DBS_2x1.6 sample type replicas with the WB_ref sample type, i.e. replica_1 vs WB_ref, replica_2 vs WB_ref and replica_3 vs WB_ref yielded similar concordance rates (see S1 Fig). Based on this, we decided to include all of the data of the DBS_2x1.6 sample type for calculation of the concordances displayed in Fig 3; that is the DBS_2x1.6 vs WB_ref and DBS_2x3.2 vs DBS_2x1.6 comparisons, respectively. This was done by initial comparison of each of the DBS_2x1.6 sample type replicas with the WB_ref or DBS_2x3.2 sample types, followed by the calculation of average values hereof. An initial look at the concordances in Fig 3 before filtering show strong dependency by variant type with SNPs > insertions > deletions > mulitiallelic calls, respectively (upper panels). This is similar to what has previously been observed in WES studies splitting calls by variant type, and results due to well-known difficulties of calling high-complexity variants as well as a general higher probability of encountering sequence variation between any two samples for variants of increasing lengths [26]. The pre-filtered concordance rates are consistent with other exome sequencing experiments dealing with sample replicas: previous reported rates were in the range of 82–92%, the numbers being largely dependent on the depth of coverage, as is also reported here [27]. Notably, we observe somewhat lower concordance rates of the DBS vs WB comparisons (i.e. all comparisons of neonatal DBS samples and WB reference samples) over the WB vs WB comparisons (all comparisons of WB reference samples) before filtering in Pilot 2 and Pilot 3, which may be ascribed to subtle differences in overall DNA quality of DBS-derived wgaDNA as compared to WB-derived DNA. This difference become neutralized after filtering yielding similar concordance rates between the DBS vs WB and WB vs WB comparisons independent of whether 2x3.2 mm or 2x1.6 mm discs were used for DNA extraction. Moreover, post-filtering the concordance rates of all pairwise compared samples are very close to 100% (the multiallelic calls of Pilots 1 and 3 omitted, for which rates only reaches 85–88%). This shows that DBS-derived wgaDNA no matter the input quantities, i.e. 2x3.2 mm or 2x1.6 mm discs can be set to perform similarly to high-quality DNA from a WB reference sample, if in case the proper filter settings are applied. This validates the use of DNA of neonatal DBSs for WES. The one most striking example illustrating the feasibility of sequencing the neonatal DNA can be inferred from the WB_ref vs WB_ref replica and DBS_2x3.2 vs WB_ref comparisons of Pilot 2. The fact that duplicate sequencing data produced using the same high-quality DNA (e.g. the WB_ref vs WB_ref replica comparison), scores similarly to that DNA set against wgaDNA of a neonatal DBS sample, proves the validity of the latter. One concern of using wgaDNA of neonatal DBS samples has been if the amplification reaction would introduce sequence errors and allele dropouts caused by the low inputs and presumed low quality of the DNA used. However, based on the very high similarities observed between the DBS vs WB and WB vs WB comparisons along with the very high concordances overall post-filtering, we conclude that this does not seem to constitute a problem.

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