Table 4.
Comparison of CFTR Clinical Sequencing Assays
| Study | NGS assay | CFTR target size | Sample type (DNA amount) | DBS | Sensitivity, % | Specificity, % | Precision, % | Reagent cost | Effort | Limitations in NBS |
|---|---|---|---|---|---|---|---|---|---|---|
| Abou Tayoun et al12 | AmpliSeq custom panel with two separate PCR primer pools, Ion torrent sequencing | CDS, 10,343 bp | 79 peripheral blood samples and Coriell cell lines (20 ng) | No | 98.6 | 97 | 100 | $154 and $103 per sample when 12 and 35 samples, respectively, are pooled per run | NA | Homopolymer sequencing errors with 2184delA false-positive calls; poly-TG and polyT tract not reported |
| Trujillano et al11 | NimbleGen SeqCap EZ Choice array, Illumina HiSeq2000 | Entire CFTR genomic locus, 182 Kbp | 92 peripheral blood samples of CF patients and carriers (1.1 μg) | No | 100 | 100 | 91 | $200 per sample | ∼14 days' TAT | High DNA sample requirement, and limited multiplexing of 24 samples |
| Grosu et al13 | Illumina TruSeq Custom Amplicon, MiSeqDx Cystic Fibrosis Clinical Sequencing Assay | CDS, two deep intronic variants and two large deletions | 366 peripheral blood samples and cell lines (250 ng) | No | 99.68 | 100 | 99.7 | $220 per sample if 48 samples are pooled (∼$1320 per sample if 8 samples are pooled∗) | <3 hours; hands-on time based on Illumina Data sheet | High 250 ng DNA sample requirement; MiSeqDX special instrumentation |
| Bonini et al37 | NimbleGen SeqCap EZ kit, or long-range PCR, 454 GS Junior Sequencer | Entire CFTR genomic locus, 188 Kbp | 18 peripheral blood samples (500 ng) | No | NA | NA | High 500 ng DNA sample requirement, and high error rate in homopolymeric polypyrimidine tract base calling | |||
| Baker et al15 | Illumina TruSeq Custom Amplicon, MiSeqDx Cystic Fibrosis Clinical Sequencing Assay | CDS, two deep intronic variants and two large deletions | 232 DBS samples (1 × 3.2 mm DBS punch) | Yes | 100 | 100 | ∼220 per sample if 48 samples are pooled (∼$1320 per sample if eight samples are pooled∗) | ∼1 week TAT, <3 hours; hands-on time based on Illumina data sheet | Investigator-use-only mode limited to the sequence analysis of 162 CFTR mutations; MiSeqDx special instrumentation | |
| Loukas et al42 | Multiplicom MASTR version 2 multiplex PCR, Illumina MiSeq | CDS, selected intronic regions and promotor | 188 peripheral blood samples and 12 DBS | Yes | ∼$300 per sample when 10 samples are pooled per run | ∼1-week TAT | Limited multiplexing of up to 20 samples/run; no CNV analysis provided | |||
| Lefterova et al (this study) | Lab-developed multiplex PCR, Illumina MiSeq | CDS, two deep intronic variants, 16,513 bp | 193 DBS samples (1 × 3.2 mm punch, ∼1 ng/sample) | Yes | 100 | 100 | 100 | ∼$15 per sample when 95 samples are pooled per run | ∼7 hours; hands-on time, ∼3 days' TAT |
Characteristics of recently developed targeted NGS assays that enable CFTR sequence analysis and detection of common CNVs.
CDS, coding sequence; CF, cystic fibrosis; CNV, copy number variant; DBS, dried blood spot; NA, not available; NBS, newborn screening; NGS, next-generation sequencing; polyT, poly-thymidine; TAT, turnaround time; TG, thymidine-guanine.