Translating Sanger-Based Routine DNA Diagnostics into Generic Massive Parallel Ion Semiconductor Sequencing

Diekstra, Adinda; Bosgoed, Ermanno; Rikken, Alwin; Lier, Bart van; Kamsteeg, Erik‐Jan; Tychon, Marloes; Derks, Ronny; Soest, Ronald A. van; Mensenkamp, Arjen R.; Scheffer, Hans; Neveling, Kornelia; Nelen, Marcel

doi:10.1373/clinchem.2014.225250

Cited by 25 publications

(20 citation statements)

References 29 publications

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“…Sequencing depth was arbitrarily chosen to resemble current clinical practice at the Radboud university medical center and other diagnostic laboratories (21)(22)(23)(24). Sequencing depths of 100ϫ, 70ϫ, and 30ϫ were assumed for TGP, WES, and WGS, respectively.…”

Section: Framework For Cost Calculationmentioning

confidence: 99%

Is the $1000 Genome as Near as We Think? A Cost Analysis of Next-Generation Sequencing

Nimwegen¹,

et al. 2016

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BACKGROUND:The substantial technological advancements in next-generation sequencing (NGS), combined with dropping costs, have allowed for a swift diffusion of NGS applications in clinical settings. Although several commercial parties report to have broken the $1000 barrier for sequencing an entire human genome, a valid cost overview for NGS is currently lacking. This study provides a complete, transparent and up-to-date overview of the total costs of different NGS applications.

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Section: Framework For Cost Calculationmentioning

confidence: 99%

Is the $1000 Genome as Near as We Think? A Cost Analysis of Next-Generation Sequencing

Nimwegen¹,

et al. 2016

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“…15 These studies used digital melt-curve analysis and pyrosequencing to detect mutations but next-generation sequencing (NGS) is being widely used in clinical laboratories to detect somatic mutations in cancer tissues. 1617 …”

Section: Introductionmentioning

confidence: 99%

Digital next-generation sequencing identifies low-abundance mutations in pancreatic juice samples collected from the duodenum of patients with pancreatic cancer and intraductal papillary mucinous neoplasms

Sadakari

Shindo

et al. 2016

Gut

141

126

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Objective Secretin-stimulated pancreatic juice contains DNA shed from cells lining the pancreatic ducts. Genetic analysis of this fluid may form a test to detect pancreatic ductal neoplasia. Design We employed digital next-generation sequencing (‘digital NGS’) to detect low-abundance mutations in secretin-stimulated juice samples collected from the duodenum of subjects enrolled in Cancer of the Pancreas Screening studies at Johns Hopkins Hospital. For each juice sample, digital NGS necessitated 96 NGS reactions sequencing nine genes. The study population included 115 subjects (53 discovery, 62 validation) (1) with pancreatic ductal adenocarcinoma (PDAC), (2) intraductal papillary mucinous neoplasm (IPMN), (3) controls with non-suspicious pancreata. Results Cases with PDAC and IPMN were more likely to have mutant DNA detected in pancreatic juice than controls (both p<0.0001); mutant DNA concentrations were higher in patients with PDAC than IPMN (p=0.003) or controls (p<0.001). TP53 and/or SMAD4 mutations were commonly detected in juice samples from patients with PDAC and were not detected in controls (p<0.0001); mutant TP53/SMAD4 concentrations could distinguish PDAC from IPMN cases with 32.4% sensitivity, 100% specificity (area under the curve, AUC 0.73, p=0.0002) and controls (AUC 0.82, p<0.0001). Two of four patients who developed pancreatic cancer despite close surveillance had SMAD4/TP53 mutations from their cancer detected in juice samples collected over 1 year prior to their pancreatic cancer diagnosis when no suspicious pancreatic lesions were detected by imaging. Conclusions The detection in pancreatic juice of mutations important for the progression of low-grade dysplasia to high-grade dysplasia and invasive pancreatic cancer may improve the management of patients undergoing pancreatic screening and surveillance.

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“…As a consequence, the cost ($1.50) per extract is ten times higher than with SMRT analysis. Diekstra et al (10) employed the Ion Torrent PGM platform to characterize up to 900 bp templates, but the amplicons had to be sheared to <300 bp before sequencing and reassembly. The resulting sequences corresponded closely with their Sanger counterparts, but analytical costs ($1.96) were only three-fold less than those for Sanger analysis ($6.00).…”

Section: Discussionmentioning

confidence: 99%

“…These limitations are an important constraint in three contexts: de novo genome assemblies are difficult (4), complex regions of well-known genomes can be intractable (5), and sequencing long amplicons is inefficient. Because of the latter constraint, Sanger sequencing is still widely used for amplicon characterization (6-9) despite its relatively high cost (10). While recent studies have established that Illumina (11,12) and Ion Torrent (10) platforms can analyze 1 kb amplicons with good accuracy, their need to concatenate short reads creates risks to data quality linked to the recovery of chimeras and pseudogenes.…”

Section: Introductionmentioning

confidence: 99%

A Sequel to Sanger: Amplicon Sequencing That Scales

Hebert

Braukmann

Prosser

et al. 2017

Preprint

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Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. By examining templates from more than 5,000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL reduces costs 40-fold from Sanger analysis. Reflecting the capacity of each instrument to recover sequences from more than five million DNA extracts a year, this platform facilitates massive amplicon characterization.

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Translating Sanger-Based Routine DNA Diagnostics into Generic Massive Parallel Ion Semiconductor Sequencing

Cited by 25 publications

References 29 publications

Is the $1000 Genome as Near as We Think? A Cost Analysis of Next-Generation Sequencing

Is the $1000 Genome as Near as We Think? A Cost Analysis of Next-Generation Sequencing

Digital next-generation sequencing identifies low-abundance mutations in pancreatic juice samples collected from the duodenum of patients with pancreatic cancer and intraductal papillary mucinous neoplasms

A Sequel to Sanger: Amplicon Sequencing That Scales

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