Elsevier

Molecular Genetics and Metabolism

Volume 110, Issues 1–2, September–October 2013, Pages 65-72
Molecular Genetics and Metabolism

Archived neonatal dried blood spot samples can be used for accurate whole genome and exome-targeted next-generation sequencing

https://doi.org/10.1016/j.ymgme.2013.06.004Get rights and content

Highlights

  • We compare whole-blood DNA NGS results to results from DBSS

  • NGS of neonatal DBSS is highly comparable to NGS of high-quality DNA

  • Whole-genome amplification of DBSS DNA does not compromise the NGS accuracy

  • Two 3.2 mm disks from a DBSS is sufficient for WGA, and subsequently WES and WGS

  • DBSS can be used for NGS, expanding their use in neonatal screening and research

Abstract

Dried blood spot samples (DBSS) have been collected and stored for decades as part of newborn screening programmes worldwide. Representing almost an entire population under a certain age and collected with virtually no bias, the Newborn Screening Biobanks are of immense value in medical studies, for example, to examine the genetics of various disorders. We have previously demonstrated that DNA extracted from a fraction (2 × 3.2 mm discs) of an archived DBSS can be whole genome amplified (wgaDNA) and used for accurate array genotyping. However, until now, it has been uncertain whether wgaDNA from DBSS can be used for accurate whole genome sequencing (WGS) and exome sequencing (WES).

This study examined two individuals represented by three different types of samples each: whole-blood (reference samples), 3-year-old DBSS spotted with reference material (refDBSS), and 27- to 29-year-old archived neonatal DBSS (neoDBSS) stored at − 20 °C in the Danish Newborn Screening Biobank. The reference samples were genotyped using an Illumina Omni2.5M array, and all samples were sequenced on a HighSeq2000 Paired-End flow cell. First, we compared the array single nucleotide polymorphism (SNP) genotype data to the single nucleotide variation (SNV) calls from the WGS and WES SNV calls. We also compared the WGS and WES reference sample SNV calls to the DBSS SNV calls.

The overall performance of the archived DBSS was similar to the whole blood reference sample. Plotting the error rates relative to coverage revealed that the error rates of DBSS were similar to that of their reference samples. SNVs called with a coverage < × 8 had error rates between 1.5 and 35%, whereas the error rates of SNVs called with a coverage  8 were < 1.5%. In conclusion, the wgaDNA amplified from both new and old neonatal DBSS perform as well as their whole-blood reference samples with regards to error rates, strongly indicating that neonatal DBSS collected shortly after birth and stored for decades comprise an excellent resource for NGS studies of disease.

Introduction

The opportunity to perform extensive genotyping on DNA extracted from dried blood spot samples (DBSS) used in the newborn screening programmes has opened new avenues in newborn screening as well as for the study of the genetic influence of many complex disorders [1], [2], [3], [4]. Residual newborn DBSS stored in national repositories combined with relevant clinical information from national medical registries provide access to large cohorts of well-characterised patients and healthy controls [5]. However, the major challenge using the DBSS for such ventures is the small amount of blood available. In theory, the amount of genomic DNA (gDNA) that can be extracted from a 3.2 mm punch of DBSS is approximately 60 ng [6]. This shortage of DNA may be overcome by whole genome amplified (wga) DNA (wgaDNA). wgaDNA amplified from DBSS stored at − 20 °C/− 4 °F for three decades in the Danish Newborn Screening Biobank (DNSB) has been shown to provide highly reliable genotyping results by different approaches: Sanger sequencing [7], genome wide arraying [1], [8], high-resolution melting curve analysis [7], and simple SNP genotyping [3]. The excess DBSS material from the Danish Newborn Screening programme has been stored in the DNSB [5] since 1982 and covers close to 100% of the Danish population born since that time. Combined with data from numerous Danish registries, the DBSS has been used successfully in several genetic studies of disease looking at cerebral palsy [9], schizophrenia [10], [11], [12], [13], [14], [15], [16], birth weight [17], psychosis [18], [19], and infantile hypertrophic pyloric stenosis [20]. In many countries and states, the neonatal screening programmes are storing the excess DBSS in biobanks, if allowed by law. In contrast to the DNSB and the California Research-Ready Biospecimen Bank, the majority of these biobanks, for example, the Michigan neonatal biobank, and the Swedish PKU-Biobank, store their DBSS at room temperature and not at − 20 °C/− 4 °F, which can complicate their use in some approaches due to degradation of the DNA. However, the potential of using the millions of already stored neonatal DBSS for research on different early onset diseases is enormous and is likely to improve the screening of newborns in the future, and to increase our biological knowledge about many diseases.

Next-generation sequencing (NGS) is currently a rapidly advancing technology within the field of genetics and is moving towards reasonable prices per sample in increasingly fast increments. Coupled with advances in data handling and analysis, this technology is on a path to becoming a standard tool in research and clinical genetics. In addition, this sequencing technology has prodigious potential for disease diagnostics and in the screening of newborns. The sensitivity of the technology is rapidly improving, with current standardised off the shelf protocols allowing the use of nanograms of high-quality input DNA for whole-genome sequencing (WGS), although larger amounts are still necessary for targeted sequencing approaches such as whole-exome sequencing (WES). A solution to the low concentrations of DNA obtainable from DBSS could be whole-genome amplification; however, it is uncertain whether the amplification process compromises the results and introduces genotype call errors or more serious de-novo mutations.

This proof-of-principle study investigates whether it is possible to use only two 3.2 mm discs from archived DBSS for DNA extraction, WGA, and subsequent WES and WGS, using standard protocols. Moreover, by comparing different sample types we aim to evaluate the crude error rates of the WES and WGS single nucleotide variation (SNV) calls by simple comparisons of called variations without using any data optimising steps or algorithms.

Section snippets

Study overview and sample preparations

The study included two individuals (siblings); A (male) and B (female). As defined by the “Danish Act on Research Ethics Review of Health Research Projects” Section 2, this project does not constitute a health research project but is considered a quality developmental project for newborn screening. The project can thus be conducted without approval from the Committees on Biomedical Research Ethics for the Capital Region of Denmark.

From each of the two adult individuals, a venous blood sample

Array and NGS evaluation

To have an accurate “gold-standard” SNP genotype for estimating the error rate of the NGS SNV calls, we genotyped the Ref. sample of individuals A and B with the HumanOmni2.5-Quad BeadChip array. The call rates of individual A and B's HumanOmni2.5-Quad BeadChip runs were 99.8521% and 99.7345%, respectively.

One lane of sequencing was generated for each WGS sample. Following this setup, the variation in absolute read counts was determined only by the achieved cluster count in each lane, which

Conclusions

This study is the first to use neonatal DBSS for NGS. Using two 3.2 mm discs from an archived neonatal DBSS, we extracted DNA, performed WGA, and used this wgaDNA for WGS and WES. The accuracy of the SNV genotyping of the DBSS wgaDNA was highly comparable to the accuracy of genotyping using the unamplified high-quality DNA samples. In conclusion, we found that reliable WGS and WES can be conducted on archived neonatal DBSS using only a fraction of the accessible material. This further adds to

Conflict of interests

The authors declare no competing interests.

Authors' contributions

MH initiated and designed the study, handled the DBSS and the adult whole-blood samples, analysed array and NGS data, interpreted the datasets and drafted the manuscript. JG prepared the samples for WES and WGS, aligned the NGS data and called the SNVs in CASAVA, and revised the manuscript. RN and SM helped designing the study, were involved in the interpretation of the data and revised the manuscript. JA helped analysing the data, was involved in the interpretation of data and revised the

Acknowledgments

We would like to acknowledge research technician Høgni Kallehauge Petersen and senior technician Lis Vestergaard Hansen for their excellent work in identifying and handling the DBSS.

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