Whole exome and whole genome sequencing with dried blood spot DNA without whole genome amplification

Bassaganyas, Laia; Freedman, George; Vaka, Dedeepya; Wan, Eunice; Lao, Richard; Chen, Flavia; Kvale, Mark N.; Currier, Robert; Puck, Jennifer M.; Kwok, Pui‐Yan

doi:10.1002/humu.23356

Cited by 38 publications

(37 citation statements)

References 9 publications

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“…Dried blood spots are potential resources for genetic and genomic analysis. Recent studies showed that sufficient DBS DNA can be extracted and used for NGS to perform whole exome sequencing (WES) and whole genome sequencing (WGS) without genome amplification ( 45 ). The use of next-generation sequencing has the potential to be integrated in SCID NBS programs to facilitate and accelerate genetic testing and final diagnosis of affected newborns.…”

Section: Discussionmentioning

confidence: 99%

High Incidence of Severe Combined Immunodeficiency Disease in Saudi Arabia Detected Through Combined T Cell Receptor Excision Circle and Next Generation Sequencing of Newborn Dried Blood Spots

et al. 2018

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Severe combined immunodeficiency disease (SCID) is the most severe form of primary immunodeficiency disorders (PID). T-cell receptor excision circle (TREC) copy number analysis is an efficient tool for population-based newborn screening (NBS) for SCID and other T cell lymphopenias. We sought to assess the incidence of SCID among Saudi newborn population and examine the feasibility of using targeted next generation sequencing PID gene panel (T-NGS PID) on DNA isolated from dried blood spots (DBSs) in routine NBS programs as a mutation screening tool for samples with low TREC count. Punches from 8,718 DBS collected on Guthrie cards were processed anonymously for the TREC assay. DNA was extracted from samples with confirmed low TREC count, then screened for 22q11.2 deletion syndrome by real-time polymerase chain reaction and for mutations in PID-related genes by T-NGS PID panel. Detected mutations were confirmed by Sanger sequencing. Sixteen out of the 8,718 samples were confirmed to have low TREC copy number. Autosomal recessive mutations in AK2, JAK3, and MTHFD1 were confirmed in three samples. Two additional samples were positive for the 22q11.2 deletion syndrome. In this study, we provide evidence for high incidence of SCID among Saudi population (1/2,906 live births) and demonstrate the feasibility of using T-NGS PID panel on DNA extracted from DBSs as a new reliable, rapid, and cost-effective mutation screening method for newborns with low TREC assay, which can be implemented as part of NBS programs for SCID.

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Section: Discussionmentioning

confidence: 99%

High Incidence of Severe Combined Immunodeficiency Disease in Saudi Arabia Detected Through Combined T Cell Receptor Excision Circle and Next Generation Sequencing of Newborn Dried Blood Spots

et al. 2018

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“…Addition of new diseases to NBS panels can be expected to reveal both anticipated information on disease incidence, spectrum and characteristics and unanticipated findings that challenge medical experts and engage basic scientists. Moreover, storage of residual DBS material collected for NBS opens the possibility of conducting much more far‐reaching research, while with modern DNA sequencing technology one can sequence a person's entire genome using a small punch from a stored DBS . Controversies and legal battles claiming breaches of the public trust have resulted in destruction of state biobanks of newborn DBS.…”

Section: Screening For Scid As a Public Health Measurementioning

confidence: 99%

“…Moreover, storage of residual DBS material collected for NBS opens the possibility of conducting much more far-reaching research, while with modern DNA sequencing technology one can sequence a person's entire genome using a small punch from a stored DBS. 21 Controversies and legal battles claiming breaches of the public trust have resulted in destruction of state biobanks of newborn DBS.…”

Section: Screening For Scid a S A Pub Lic He Alth Me A Surementioning

confidence: 99%

Newborn screening for severe combined immunodeficiency and T‐cell lymphopenia

Puck

2018

Immunological Reviews

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134

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Summary The development of a T cell receptor excision circle (TREC) assay utilizing dried blood spots made possible universal newborn screening (NBS) for severe combined immunodeficiency (SCID) as a public health measure. Upon being flagged by an abnormal screening test in a SCID screening program, an infant can receive further diagnostic testing for SCID in the neonatal period, prior to onset of infectious complications, to pemit immediate institution of protective measures and definitive, life-saving treatment to establish a functional immune system. SCID screening is now the accepted standard of care in state public health departments across the USA, and it is being adopted in many countries. It has proven effective, with infants having this otherwise inapparent but serious, rare disorder achieving survival and immune reconstitution. In addition to bringing to attention infants with the primary screening target diseases, typical SCID and leaky SCID (due to hypomorphic mutations in known SCID genes), the newborn screening assay for insufficient TRECs in dried blood spots also reveals infants with non-SCID T lymphopenic conditions. Experience has accumulated regarding the range and limitations of diagnoses of newborns with low TRECs and low T cells. Previously unknown immune defects have been discovered, as well as conditions not formerly recognized to have low T cells in the neonatal period.

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“…DNA extraction, library preparation and sequencing were carried out following a previously described protocol. (Bassaganyas, et al, 2018) The reads from the NBSeq exomes were mapped to the human reference genome GRCh37 assembly (Aug 2009 release), using BWA-mem v0.7.10 ( Li and Durbin, 2009). The resulting BAM files were sorted, indexed and marked for PCR duplicate reads by Picard v0.7.10 (http://picard.sourceforge.net).…”

Section: Exome Sequencing and Analysismentioning

confidence: 99%

Perturbation robustness analyses reveal important parameters in variant interpretation pipelines

Wang¹,

Adhikari²,

Sunderam

et al. 2020

Preprint

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AbstractMotivationGenome sequencing is being used routinely in clinical and research applications, but subsequent variant interpretation pipelines can vary widely. A systematic approach for exploring parameter choices and selection plays an important role in designing robust pipelines for specific clinical applications.ResultsWe present a framework to be applied in scenarios with limited data whereby expert knowledge informs pipeline refinement. Starting from initial reference variant interpretation pipelines with commonly used parameters, we derived pipelines by perturbing the parameters one by one to determine which parameters can yield meaningful changes in a pipeline’s performance. We updated the reference pipeline by fixing the value of parameters which have small impact on the pipeline’s performance. Then we conducted new rounds of perturbation as the process converged, yielding a stable pipeline which is robust. We applied the framework for genetic disease prediction in de-identified exomes from a cohort of 138 individuals with rare Mendelian inborn errors of metabolism (IEMs) and systematically explored how perturbing different parameters affected the pipeline’s sensitivity and specificity. For this application, we perturbed commonly used parameters in variant interpretation pipelines, including choices of genes, variant callers, transcript models, databases of allele frequencies, databases of curated disease variants, and tools for variant impact prediction. Our analyses showed that choice of variant callers, variant impact prediction tools, MAF threshold, and MAF databases can meaningfully alter results from a pipeline. This work informs the development of exome analysis pipelines designed for newborn metabolic disorder screening and suggests the general application of perturbation analysis in genome interpretation pipeline design.

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Whole exome and whole genome sequencing with dried blood spot DNA without whole genome amplification

Cited by 38 publications

References 9 publications

High Incidence of Severe Combined Immunodeficiency Disease in Saudi Arabia Detected Through Combined T Cell Receptor Excision Circle and Next Generation Sequencing of Newborn Dried Blood Spots

High Incidence of Severe Combined Immunodeficiency Disease in Saudi Arabia Detected Through Combined T Cell Receptor Excision Circle and Next Generation Sequencing of Newborn Dried Blood Spots

Newborn screening for severe combined immunodeficiency and T‐cell lymphopenia

Perturbation robustness analyses reveal important parameters in variant interpretation pipelines

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