Table 3.

Comparison of Method Input DNA Required, Mutations Detected, Number of Reactions, Run Time, and Run Capacity.^a

Method	No. Mutations/ Variants Detected	No. Reactions	DNA Volume Used in Studyb	Avgerage DNA Qty Used Per Reaction (Min- Max)b	Library Prep Time	PCR Setup and Run Time	Post-PCR Processing/ Run Time	Instrument Run Time	Total Run Time	Single-Run Capacityc	System Dedi- cated Software	Mutation Report
Sanger	Unlimited in regions amplified	42PCR rxns86 cycle sequencing	42 µL(I µL/ PCR rxn)	5–10 ng	NA	3 hours	4 hours	14.5 hours	21.5 hours	Nine 384-well plates (+2, 96-well plates with varying run conditions)	Y SeqScape	Does not distinguish variants from defined mutations—requires expert interpretation
Ion AmpliSeq CFTR Community Panel	Unlimited in regions amplified	2 Pools	12 µL (6 µL/ pool)	18.2 ng (0.76–8.98 ng/µL)	4–6 hours	NA	6–7 hours	2–7 hours (dependent on chip size)	12–20 hours	Eight 314 chip (500 ×) Forty-nine 316 chip (500 ×) Ninety-six 318 chip (500 ×)	Y Coverage Analysis Variant Caller	CFTR2 defined mutations and variants listed in Hotspot file are annotated (Legacy and HGVS)—novel mutations and variants require expert interpretation
Swift Biosciences Accel- Amplicon CFTR Panel	Unlimited in regions amplified	1 Pool	5–10 µL	19.3 ng (0.55– 5.21 ng/µL)	2.5 hours	NA	1.5 hours	28 hours	32 hours	Forty-eight Nano flow cell (400×) Forty-eight Micro flow cell (1600×)	Nd Freeware: Cutadapt, BWA-MEM, FreeBayes, GATK	Does not distinguish variants from defined mutations—requires expert interpretation
MiSeqDx Cystic Fibrosis 139-Variant Assay	139	1 Pool	5 µL	8.95 ng (0.55– 3.88 ng/µL)	5 hours	NA	3 hours	28 hours	36 hours	Forty-eight flow cells	Y MiSeq Reporter	Defined mutations identified
Hologic InPlex CF Molecular Test 40+4	42 + 2 variants	1 rxn	5 µL	Not quantified	NA	2.5 hours	1 hour	5 minutes	3.5 hours	8 Cards	Y Call Reporting Software	Defined mutations identified
Luminex xTAG CF39v2 kit	39+4 variants	1 rxn	5 µL	Not quantified	NA	2 hours	3.5 hours	1 hour	6.5 hours	Ninety-six 96- well plates	Y TDAS CFTR	Defined mutations identified
Luminex xTAG CF60v2 kit	60+4 variants	1 PCR rxns 2 ASPE rxns	5 µL	Not quantified	NA	2 hours	4 hours	1 hour	7 hours	Forty-eight 96- well plates	Y TDAS CFTR	Defined mutations identified
Thermo Fisher Scientific TaqMan SNP Genotyping	44	45 rxns	45 µLe (1 µL/mutation)	1.59 ng (0.62– 4.82 ng/µL)	NA	15 minutes - 96-well plate 30 minutes - 384-well plate 30 minutes - OpenArray plate	NA	1.5 hours	~2 hours - 96- well plate ~2 hours - 384-well plate ~4 hours - OpenArray plate	Two 96-well plates Eight 384- well plate Forty-eight OpenArray plates (64 assay format)	Y Genotyper	Defined mutations identified—some mutations require interpretation using multiple probes (intron 9-5/7/9T and I507del and F508del region)

Abbreviations: ASPE, allele specific primer extension; CF, cystic fibrosis; CFTR, CF transmembrane regulator; DBS, dried blood specimen; HGVS, human genome variation society; NA, not available; NBS, newborn screening; PCR, polymerase chain reaction; rxn, reaction; SNP, single-nucleotide polymorphism.

^aThe run times in this table are based on our laboratory’s experience, however it does not include analysis of the data. The Bioinformatics are described in the materials and methods.

^bTypically DNA is not quantitated prior to use in NBS labs, so some of the quantities are estimated based on average known concentration of DNA extracted from adult DBS.

^cThe single-run capacity indicates the number of samples that can be loaded onto an instrument at a time.

^dThe Accel-Amplicon CFTR panel is still in development, the company plans on offering a Bioinformatic pipeline (T. Harkins, personal communication, March 29, 2016).

^eOpenArray was not performed in this study. The manufacturer recommended input is 2.5 µL of DNA exctracted from DBS.