Abstract
Next generation DNA sequencing (NGS) has the potential to improve the diagnostic and prognostic utility of newborn screening programmes. This study assesses the feasibility of automating NGS on dried blood spot (DBS) DNA in a United Kingdom National Health Service (UK NHS) laboratory. An NGS panel targeting the entire coding sequence of five genes relevant to disorders currently screened for in newborns in the UK was validated on DBS DNA. An automated process for DNA extraction, NGS and bioinformatics analysis was developed. The process was tested on DBS to determine feasibility, turnaround time and cost. The analytical sensitivity of the assay was 100% and analytical specificity was 99.96%, with a mean 99.5% concordance of variant calls between DBS and venous blood samples in regions with ≥30× coverage (96.8% across all regions; all variant calls were single nucleotide variants (SNVs), with indel performance not assessed). The pipeline enabled processing of up to 1000 samples a week with a turnaround time of four days from receipt of sample to reporting. This study concluded that it is feasible to automate targeted NGS on routine DBS samples in a UK NHS laboratory setting, but it may not currently be cost effective as a first line test.
Highlights
Newborn screening (NBS) programmes worldwide provide early diagnosis and intervention for infants affected with rare disorders which, if untreated, are associated with progressive deterioration, disability and potentially fatal outcomes [1]
We used the Ion Torrent S5 XL platform, as the rapid amplicon-based library preparation enables a fast turnaround time; it has the potential for good cost effectiveness and it affords a high degree of sample multiplexing and, throughput
There was one small exonic region that had an average coverage below 30× and another that had an average coverage between 30 and 50× (CFTR exon 1), with all other regions having an average coverage of ≥50× (Figure S4)
Summary
Newborn screening (NBS) programmes worldwide provide early diagnosis and intervention for infants affected with rare disorders which, if untreated, are associated with progressive deterioration, disability and potentially fatal outcomes [1]. DNA testing is only undertaken as a second line test in the NBS protocol for cystic fibrosis, as part of the follow-up diagnostic protocol for suspected cases of medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), isovaleric acidaemia (IVA) and glutaric aciduria type 1 (GA1) and for further characterisation of a subset of suspected haemoglobinopathy cases [3,6,9]. The advent of generation sequencing (NGS) has resulted in attempts to expand the use of DNA sequencing in NBS to improve diagnostic and prognostic utility [10,11,12,13]. Several key features of NGS make it a potentially powerful technology in NBS. The increasing trend toward provision of genetic laboratory services in larger centralised units in the UK has the potential to enable greater access to high throughput NGS technology. It is timely and relevant to investigate the potential of NGS in NBS in a UK NHS context
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