Table 1 Analytic performance, reproducibility, and duration of the major steps in automated diagnosis of genetic diseases by accelerated rWGS.
Sample | 362 | 12878 | NA24385 | AG928 | AG366 | AF414 | AI003 | AH638 | CSD59F | CSD709 | ||||||||
Run | Ref. 16 | 927 | 929 | 930 | 1018 | 1020 | 1204 | 1208 | 1218 | 1026 | 1027 | 477 | 480 | 478 | 479 | |||
Sample & Run Type | DNA/Analytic performance | Blood/Retrospective | Blood/Prospective | |||||||||||||||
Diagnosis (Gene) | None | None | None | ALDOB | OTC | PCCA | SCN2A | SLC19A3 | MT-ATP6, SETD1A | ADAMTSL2 | ||||||||
rWGS Methods | Ref 16 | Herein | Herein | Here | Std | Here | Std | Here | Std | |||||||||
SV & CNV ID Method | None |
| MC |
| MC |
| MC |
|
|
|
|
|
| D3.5 |
| D3.5 |
| D3.5 |
Length of steps (min) | ||||||||||||||||||
Sample Prep. Time | 151 | 50 | 45 | 41 | 50 | 74 | 71 | 69 | 67 | 80 | 1233 | 90 | 265 | 90 | 265 | |||
Sequencing Time | 932 | 667 | 667 | 666 | 673 | 674 | 667 | 683 | 675 | 676 | 1067 | 687 | 1050 | 687 | 1050 | |||
10 /20 Analysis Time | 62 | 48 | 191 | 45 | 181 | 46 | 194 | 48 | 42 | 55 | 37 | 38 | 47 | 173 | 44 | 185 | 56 | 220 |
Tertiary Analysis | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | n.a. | 10 | 14 | 13 | 13 | 10 | 87 | 12 | 126 | 21 | 131 |
Total Time to Result | 1145 | 765 | 903 | 757 | 888 | 753 | 917 | 761 | 800 | 807 | 802 | 793 | 812 | 2560 | 833 | 1626 | 854 | 1666 |
Sequence metrics | ||||||||||||||||||
Trimmed yield (Gigabases) | 149 | 192 | 178 | 186 | 189 | 165 | 176 | 80 | 135 | 187 | 162 | 182 | 144 | 174 | 153 | |||
Reads with quality score >30 | 90.7% | 90.5% | 88.7% | 90.8% | 91.3% | 89.2% | 91.2% | 92.5% | 87.3% | 90.5% | 92.6% | 90.9% | 89.8% | 90.1% | 89.3% | |||
Error rate | n.a. | 0.17% | 0.21% | 0.17% | 0.14% | 0.19% | 0.16% | 0.14% | 0.29% | 0.17% | 0.15% | 0.14% | 0.14% | 0.17% | 0.16% | |||
Reads mapped | 98.9% | 96.7% | 96.8% | 96.8% | 97.2% | 96.0% | 96.9% | 89.0% | 94.8% | 96.2% | 99.1% | 96.1% | 99.1% | 95.5% | 98.6% | |||
Duplicate reads | 8.5% | 11.6% | 10.8% | 12.9% | 13.9% | 15.2% | 15.5% | 23.2% | 14.5% | 13.7% | 11.4% | 15.8% | 10.4% | 14.9% | 13.6% | |||
Mean insert size (Nt) | 345 | 395 | 438 | 449 | 445 | 440 | 426 | 496 | 468 | 465 | 423 | 491 | 467 | 502 | 460.5 | |||
Average genome coverage | 47.5 | 52.3 | 49.1 | 49.9 | 50.5 | 44.5 | 47.44 | 19.46 | 36.7 | 49.1 | 45.7 | 46.9 | 40.7 | 45.1 | 41.5 | |||
MIM genes w. >10X coverage of all coding domain Nt. | 95.8% | 97.6% | 96.4% | 96.7% | 97.1% | 94.9% | 95.8% | 4.2% | 92.2% | 90.1% | 95.5% | 94.6% | 94.5% | 94.7% | 94.6% | |||
Variant metrics | ||||||||||||||||||
Nt Variants (1000 s) | 4733 | 4834 | 4838 | 4838 | 4837 | 4857 | 3789 | 3904 | 4851 | 4691 | 4690 | 4852 | 4852 | 4916 | 4910 | |||
Variants passing Quality Metrics | 96.8% | 98.9% | 99.1% | 99.1% | 99.0% | 99.0% | 99.0% | 98.4% | 98.6% | 98.9% | 98.9% | 99.0% | 98.9% | 98.9% | 98.9% | |||
Coding domain variants | 0.58% | 0.51% | 0.52% | 0.52% | 0.52% | 0.53% | 0.52% | 0.52% | 0.51% | 0.52% | 0.52% | 0.52% | 0.52% | 0.53% | 0.53% | |||
Nt insertions & deletions | 17.5% | 19.7% | 19.7% | 19.7% | 19.6% | 19.5% | 19.6% | 18.9% | 19.4% | 19.6% | 19.6% | 19.7% | 19.7% | 19.7% | 19.7% | |||
Transition/transversion ratio | 2.02 | 2.03 | 2.02 | 2.02 | 2.02 | 2.03 | 2.03 | 2.03 | 2.03 | 2.03 | 2.03 | 2.03 | 2.03 | 2.03 | 2.03 | |||
- Analytic and diagnostic reproducibility were examined for sample 362 from 19.5-h rWGS (16), reference samples NA12878 and NA24385, four retrospective samples/diagnoses (AG928/Hereditary fructose intolerance (compound heterozygous, pathogenic (P) SNVs in aldolase B [ALDOB c.448 G > C, c.524 C > A]); AG366/Ornithine transcarbamylase deficiency (hemizygous, de novo, P, SNV in ornithine transcarbamylase [OTC c.275 G > A]); AF414/Propionic acidemia (homozygous, likely pathogenic (LP) indel in α-subunit of propionyl-CoA carboxylase [PCCA c.1899 + 4_1899 + 7del]); AI003/Developmental and epileptic encephalopathy 11 (heterozygous, de novo, LP SNV in the α2-subunit of the voltage-gated sodium channel [SCN2A c.4437 G > C]), and three prospective samples (AH638/Thiamine metabolism dysfunction syndrome 2 (homozygous, P, frame-shift variant in solute carrier 19, member 3 [SLC19A3 c.597dup]), CSD59F (heteroplasmic, P, SNV in the mitochondrial ATP synthase 6 gene [MT-ATP6 m.8993 T > C]), and CSD709/ Geleophysic dysplasia (compound heterozygous SNVs in ADAMTS-like 2 [ADAMTSL2 c.338 G > T and c.1851C > A]), which received rWGS both with the 13.5-h method (Herein) and standard, singleton or trio, clinical rWGS (Std) (Table S2). Ref.:16 Reference 16. Sample 12878: Sample NA12878. ID: Identification. Here: Herein. 10/20 analysis time: Conversion of raw data from base call to FASTQ format, read alignment to the reference genomes and variant calling. Tertiary analysis: Time of automated interpretation to provisional diagnosis (most rapid of three systems run in parallel (MOON, Illumina TruSight Software Suite and GEM). SV and CNV detection methods: MC: Manta and CNVnator.
: DRAGEN version 3.7. D3.5: DRAGEN version 3.5.3. MIM: Mendelian inheritance in man. Nt: Nucleotide. Gene symbols are shown in italics. Variant section headers are shown in bold.
: DRAGEN version 3.7. D3.5: DRAGEN version 3.5.3. MIM: Mendelian inheritance in man. Nt: Nucleotide. Gene symbols are shown in italics. Variant section headers are shown in bold.