Whole-genome variant detection in long-read sequencing data from ultra-low input patient samples

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Long-read sequencing offers superior genome coverage compared to short-read methods, excelling in detecting structural variants and tandem repeats.
• High input DNA requirements have been a significant limitation for long-read sequencing applications.
• Identifying genetic variants in difficult-to-map genomic regions remains a challenge for current sequencing technologies.

Purpose of the Study:

• To evaluate the performance of amplification-based long-read whole-genome sequencing methods, specifically Ultra-Low Input HiFi (ULI-HiFi) and droplet multiple displacement amplification (dMDA).
• To benchmark the accuracy of ULI-HiFi and dMDA against established variant calls from the Genome in a Bottle consortium.
• To assess the utility of ULI-HiFi for analyzing medically relevant genes and patient samples, including those from hereditary cancer syndromes.

Main Methods:

• Comparative analysis of ULI-HiFi and dMDA sequencing methods using a reference genome dataset (NA24385).
• Benchmarking variant calling accuracy for single nucleotide variants (SNVs) and tandem repeats (TRs).
• Application of ULI-HiFi to analyze normal, polyp, and adenocarcinoma samples from a patient with familial adenomatous polyposis (FAP).

Main Results:

• ULI-HiFi demonstrated high precision and recall for SNVs (F1 score 99.82%) compared to dMDA (F1 score 89.46%).
• ULI-HiFi achieved high concordance (90.4% perfect, 98.9% with single motif difference) across over 1.6 million tandem repeats.
• ULI-HiFi successfully identified a progressively expanding tandem repeat in the 5' UTR of the tumor suppressor gene *LIMD1* in FAP patient samples.

Conclusions:

• ULI-HiFi sequencing significantly improves the accuracy and efficiency of long-read whole-genome sequencing, especially with low DNA input (10-20 ng).
• This method enhances the characterization of genetic variants in previously inaccessible ('dark') genomic regions.
• ULI-HiFi provides valuable insights into the genetic basis of diseases like familial adenomatous polyposis by revealing complex variant patterns.