December 14, 2023

Beyond the Exome: Utility of Long‑Read Whole Genome Sequencing in Exome‑Negative Autosomal Recessive Diseases

Lama AlAbdi, Hanan E Shamseldin, Ebtissal Khouj, Rana Helaby, Bayan Aljamal, Mashael Alqahtani, Aisha Almulhim, Halima Hamid, Mais O Hashem, Firdous Abdulwahab, Omar Abouyousef, Amal Jaafar, Tarfa Alshidi, Mohammed Al-Owain, Amal Alhashem, Saeed Al Tala, Arif O Khan, Elham Mardawi, Hisham Alkuraya, Eissa Faqeih, Manal Afqi, Salwa Alkhalifi, Zuhair Rahbeeni, Samya T Hagos, Wijdan Al-Ahmadi, Seba Nadeef, Sateesh Maddirevula, Khalid S A Khabar, Alexander Putra, Angel Angelov, Changsook Park, Ana M Reyes-Ramos, Husen Umer, Ikram Ullah, Patrick Driguez, Yoshinori Fukasawa, Ming Sin Cheung, Imed Eddine Gallouzi, Fowzan S Alkuraya.

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 LongRead 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.  

Beyond the exome: utility of long-read whole genome sequencing in exome-negative autosomal recessive diseases

Tabular summary of the solved cases by lrWGS and identified variants: 


Novel diagnosis established by lrWGS 




Patient phenotype 



Structural variant (insertion) 

lactic acidosis and mitochondrial pathology 

RT-qPCR confirmed reduced expression 


Structural variant (deletion) 

Syndromic intellectual disability 

RT-qPCR confirmed reduced expression 


Structural variant (insertion) 

Retinitis pigmentosa 

Compound heterozygous: one allele inherited from affected mother; second allele inherited from unaffected father   


SNV in 5’UTR 

Non-syndromic band keratopathy 

RT-qPCR confirmed reduced expression. 


Deep intronic SNV 

Neuro-developmental disorder 

RT-qPCR confirmed reduced expression. Transmission electron microscopy confirmed fewer number of synaptic vesicles compared to control cells 


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 


4pb deletion 

Diamond-Blackfan syndrome-like 

Previously identified by srES, discarded as an incidental finding. RT-qPCR confirmed splicing defect.  



Severe bilateral polycystic kidney disease 

Previously identified by srES, discarded due to high population frequency.  



Skeletal dysplasia  

Previously identified by srES, discarded owing to zygosity mismatch between the probands.  



Non-syndromic retinal degeneration 

Previously identified by srES, discarded due to low in-silico prediction and no effect seen on RT-PCR 



throughput heart failure 

Previously identified by srES, discarded due lack of established genotype-phenotype correlation 




Previously identified by srES, discarded due to phenotypic inconsistency in the affected patient. 



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


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