Table 1 Proof of concept and sensitivity study results.

From: One in seven pathogenic variants can be challenging to detect by NGS: an analysis of 450,000 patients with implications for clinical sensitivity and genetic test implementation

Study

Interlaboratory pilot studya

Sensitivity studyb

Workflow

1A

1B

2

3

4

5

6

7A

7B

8

1B

Sequencing

IPE

IPE

IPE

IPE

IPE

IPE

IPE

IPE

IPE

Ion

IPE

Targeting

Hyb

Hyb

Hyb

Hyb

Hyb

Hyb

GS

Hyb

Hyb

Amp

Hyb

Informatics

CS

CS

CS

TP

CS

CS

TP

EV

EV

EV

CS

Samples

Syn (n = 2)

Syn (n = 2)

Syn (n = 2)

Syn (n = 2)

Syn (n = 2)

Syn (n = 2)

Syn (n = 2)

Syn (n = 2)

Syn (n = 2)

Syn (n = 2)

GIAB (n = 7)

Syn (n = 3)

Ref (n = 58)

Clinical (n = 26)

SNVs

15/15

15/15

15/15

11/11

15/15

12/12

15/15

11/11

15/15

10/10

434/434

1/1

1/1

18/18

Short Indel

11/11

11/11

11/11

10/10

11/11

11/11

11/11

9/11

10/11

5/6

36/36

22/22

43/43

8/8

Large indel

4/4

4/4

4/4

2/3

4/4

4/4

4/4

0/3

3/4

0/2

0/0

8/8

6/6

0/0

Segdup-associated

3/3

3/3

3/3

0/0

2/3

2/3

2/3

2/3

2/3

0/0

0/0

2/2

0/0

0/0

Low complexity

4/4

4/4

4/4

4/4

3/4

4/4

3/4

3/3

3/4

2/4

0/0

6/6

2/2

1/1

Complex and small CNV

2/2

2/2

2/2

0/2

0/2

0/2

1/2

0/2

1/2

0/2

0/0

3/3

3/3

2/2

CNV ≥2 exon

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

N/A

0/0

0/0

4/4

1/1

  1. Amp amplicon sequencing, CNV copy-number variant, CS custom software, EV software provided by the sequencing equipment vendor, GIAB Genome in a Bottle, GS genome sequencing, Hyb hybridization capture, IPE Illumina Paired-end, indel insertion or deletion, Ion Ion Torrent, N/A not applicable, NGS next-generation sequencing, Ref reference specimens from public biobanks, Segdup segmental duplication, SNVs single-nucleotide variants, Syn synthetic controls, TP third-party software.
  2. aFor the interlaboratory pilot study, the performance of 10 NGS workflows used by seven collaborating laboratories is shown for variants in two synthetic control specimens. In each data cell, the denominator is the number of variants within each assay’s target regions and the numerator is the number of these variants that were detected. Normal font indicates 100% observed sensitivity. Bold font indicates an observed limitation. Italics indicate that no study variants were present in regions interrogated by the assay. Details of each of the 10 workflows and the variants are provided in Tables S1 and S2. All workflows included bioinformatics methods to detect SNVs and small indels, and half (workflows 1A, 1B, 2, 6, and 7B) included additional methods to improve sensitivity for large indels and complex variants. CNVs were not included in this study. Workflow 1A had previously detected all of these variants in patients and was included primarily to validate the construction of the synthetic controls and to allow the comparison of synthetic and patient specimen data for the same variants (Figure S1). Workflow 1B corresponds to Fig. 2 and was an evolution of 1A, albeit with substantial differences in the variant calling algorithms used (Table S2, Fig. 2).
  3. bFor the sensitivity study, performance is shown for variants in samples from each source. Positive controls with technically challenging variants were difficult to obtain, requiring a large number of reference specimens and additional synthetic controls. A list of samples used in this study is provided in Table S5.
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