ama AlAbdi et al., Genome Medicine 2023
DNA sequencing and the identification of disease-causing mutations and human genetic variation has revolutionized medicine over recent decades. The widespread use and relative affordability of short-read exome sequencing (srES) and short-reads whole genome sequencing (srWGS), have led to a large number of scientific discoveries in genetics and human diseases. While extremely useful, the read length of srES, on the order of 100–300 bp per read, can result in the loss of resolution of genomic regions that are not uniquely spanned by overlapping reads of this size. This limitation is particularly true for low-complexity repetitive loci, duplicated regions, tandem arrays, and complex structural variants, which collectively make up the majority of the gaps and missing sequence. Long-reads whole genome sequencing (lrWGS) can sequence through DNA fragments that are orders of magnitude longer than the fragments sequenced using short-reads; i.e., 10–20 kbp fragments with very high accuracy and up to mega base-pairs (Mbp) with low accuracy using single-molecule, real-time (SMRT) sequencing by synthesis. Currently, the two main technologies for long reads sequencing (LRS) are Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). Despite their availability for more than a decade, their clinical use has been limited mainly by the sheer number of labs using the srES. However, recent studies have increasingly reported on the high yield and diagnostic utility of LRS over srES and srWGS.
Read more on Long-Read Sequencing:
All you need to know on Long–Read Sequencing Data Analysis From A-Z.
In the current study, lrWGS highlighted the increased genetic diagnostic yield of up to 38% in patients previously remaining undiagnosed on the srES platform.
In this study, 34 families of Middle Eastern ancestry were recruited after obtaining informed consent. Index patient in these families had phenotypes consistent with autosomal recessive etiology based on the phenotype or the family history, and a negative srES. LrWGS on high molecular genomic DNA (gDNA) obtained from the patient’s lymphoblastoid cell lines (LCL) was performed. RNA was also isolated from patients LCL in order to confirm the effect of splicing variants at RNA level. For lrWGS, first an optimal coverage threshold was established by sequencing one sample at coverage corresponding to 10X, 20X, 30X, and 40X, and subsequently all samples were sequenced on one SMRT cell that yielded an average depth of 10X which offered the best solution in terms of coverage and cost. The read-alignment (hg19), data processing, candidate variant prioritization workflow was done as per standard protocol.
Using autozygome-guided analysis of the lrWGS data, candidate variants were identified in 13 of the 34 families (38% diagnostic yield). A casual pathogenic variant was identified by the lrWGS in the TYMS, STK25, RP1L1, SLC4A4, SNAP91, LEMD2 genes which were not identified by the srES.
Tabular summary of the solved cases by lrWGS and identified variants:
Novel diagnosis established by lrWGS
| |||
Gene | Variant | Patient phenotype | Comments |
TYMS | Structural variant (insertion) | lactic acidosis and mitochondrial pathology | RT-qPCR confirmed reduced expression |
STK25 | Structural variant (deletion) | Syndromic intellectual disability | RT-qPCR confirmed reduced expression |
RP1L1 | Structural variant (insertion) | Retinitis pigmentosa | Compound heterozygous: one allele inherited from affected mother; second allele inherited from unaffected father |
SLC4A4 | SNV in 5’UTR | Non-syndromic band keratopathy | RT-qPCR confirmed reduced expression. |
SNAP91 | Deep intronic SNV | Neuro-developmental disorder | RT-qPCR confirmed reduced expression. Transmission electron microscopy confirmed fewer number of synaptic vesicles compared to control cells |
LEMD2 | Deletion variant | Complex neuro-developmental disorder | RT-qPCR confirmed reduced expression. Patient-derived fibroblasts had a dysmorphic nuclear morphology |
Novel variant interpretation established with the help of lrWGS | |||
FLVCR1 | 4pb deletion | Diamond-Blackfan syndrome-like | Previously identified by srES, discarded as an incidental finding. RT-qPCR confirmed splicing defect. |
PKHD1 | SNV | Severe bilateral polycystic kidney disease | Previously identified by srES, discarded due to high population frequency. |
BRIP1 | SNV | Skeletal dysplasia | Previously identified by srES, discarded owing to zygosity mismatch between the probands. |
STX3 | InDel | Non-syndromic retinal degeneration | Previously identified by srES, discarded due to low in-silico prediction and no effect seen on RT-PCR |
NID1 | SNV | throughput heart failure | Previously identified by srES, discarded due lack of established genotype-phenotype correlation |
ABHD12 | SNV | Developmentalregression | Previously identified by srES, discarded due to phenotypic inconsistency in the affected patient. |
C1orf109 | SNV | Neurodevelopmental disorder | Previously identified by srES, discarded by the analysis pipeline |
In this study, despite being the largest to date on autosomal recessive phenotypes cohort tested on lrWGS and yielding a high diagnostic yield (38%), number limitation were reported where lgWGS failed to identify the candidate variant; mainly in one proband with neurodevelopmental disorder where optical genome mapping revealed a casual novel insertion in the CHMP5 gene; in second proband where chromosomal microarray revealed a heterozygous duplication in the classical Split-hand/foot malformation 3 locus 10q24 in the affected proband; in another proband yielding positive results on increasing the lrWGS coverage to 50x.
This study clearly demonstrated the utilization of lrWGS in cases which remained undiagnostic after the srES. It further highlighted that increased coverage could be employed for the lrWGS if a patient still remains undiagnosed on low coverage lrWGS. lrWGS demonstrated a clear advantage in detecting SVs, an important class of variants that remain challenging for short-read sequencing even with improved bioinformatic handling of the data. While lrWGS clearly uncovers causal variants that are missed by exome, interpretation challenges remain an important etiology of non-diagnostic exomes. In addition, it is still unclear what is the increase of diagnostic yield of lrWGS over srWGS.
