Biology subjects

Liljedahl, U.

Publications and source records attributed to Liljedahl, U..

6 recordsLinked to original sources

bioRxiv2026

upSPLAT: Early-Barcoded Library Preparation for Cost-Effective Population-Scale Genomics

Advances in high-throughput sequencing have substantially reduced sequencing costs, yet library preparation remains a major financial and logistical bottleneck, particularly for high-throughput applications or low-quality DNA inputs. Here, we introduce upscaled Splinted Ligation Adapter Tagging (upSPLAT), a library preparation strategy that combines early sample barcoding with single-stranded splinted ligation to enable highly multiplexed pooled sequencing at substancially reduced cost. upSPLAT supports flexible high-plex pooling and reduces per-sample library preparation costs by approximately 10-fold compared to conventional workflows. By leveraging single-strand ligation, upSPLAT is compatible with a wide range of DNA inputs, including degraded, damaged or denatured double stranded DNA, bisulfite or enzymatically converted DNA, and viral single-stranded DNA. We present two complementary workflows and evaluate their performance across multiple species and DNA qualities, demonstrating robust demultiplexing, uniform sample representation, and low barcode cross-assignment. Together, upSPLAT provides a scalable, cost-effective solution for sequencing-based studies requiring large sample numbers while preserving individual-level information.

genomics↗

bioRxiv2021

Whole Genome and Exome Sequencing Reference Datasets from A Multi-center and Cross-platform Benchmark Study

With the rapid advancement of sequencing technologies in the past decade, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from errors or artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically benchmarking sequencing platforms, library protocols, bioinformatics pipelines and for measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples, a human triple-negative breast cancer cell line and a matched normal cell line derived from B lymphocytes. We generated whole-genome (WGS) and whole-exome sequencing (WES) data using 16 NGS library preparation protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the paired reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

bioinformatics↗

bioRxiv2020

The SEQC2 Epigenomics Quality Control (EpiQC) Study: Comprehensive Characterization of Epigenetic Methods, Reproducibility, and Quantification

Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and a global resource for epigenetics research from the FDAs Epigenomics Quality Control (EpiQC) Group. The study design leverages seven human cell lines that are designated as reference materials and publicly available from the National Institute of Standards and Technology (NIST) and Genome in a Bottle (GIAB) consortium. These samples were subject to a variety of genome-wide methylation interrogation approaches across six independent laboratories, with a primary focus was on 5-methylcytosine modifications. Each sample was processed in two or more technical replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), one enzymatic deamination method (EMseq), targeted methylation sequencing (Illumina Methyl Capture EPIC), and single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, BWAMeth, and GemBS), we found overall high concordance between assays (R=0.87-R0.93), differences in efficency of read mapping and CpG capture and coverage, and platform performance. The data provided herein can guide continued used of these reference materials in epigenomics assays, as well as provide best practices for epigenomics research and experimental design in future studies.

genomics↗

bioRxiv2019

Establishing reference samples for detection of somatic mutations and germline variants with NGS technologies

We characterized two reference samples for NGS technologies: a human triple-negative breast cancer cell line and a matched normal cell line. Leveraging several whole-genome sequencing (WGS) platforms, multiple sequencing replicates, and orthogonal mutation detection bioinformatics pipelines, we minimized the potential biases from sequencing technologies, assays, and informatics. Thus, our \"truth sets\" were defined using evidence from 21 repeats of WGS runs with coverages ranging from 50X to 100X (a total of 140 billion reads). These \"truth sets\" present many relevant variants/mutations including 193 COSMIC mutations and 9,016 germline variants from the ClinVar database, nonsense mutations in BRCA1/2 and missense mutations in TP53 and FGFR1. Independent validation in three orthogonal experiments demonstrated a successful stress test of the truth set. We expect these reference materials and \"truth sets\" to facilitate assay development, qualification, validation, and proficiency testing. In addition, our methods can be extended to establish new fully characterized reference samples for the community.

bioinformatics↗

bioRxiv2017

Data quality of Whole Genome Bisulfite Sequencing on Illumina platforms

The powerful HiSeq X sequencers with their patterned flowcell technology and fast turnaround times are instrumental for many large-scale genomic and epigenomic studies. However, assessment of DNA methylation by sodium bisulfite treatment results in sequencing libraries of low diversity, which may impact data quality and yield. In this report we assess the quality of WGBS data generated on the HiSeq X system in comparison with data generated on the HiSeq 2500 system and the newly released NovaSeq system. We report a systematic issue with low basecall quality scores assigned to guanines in the second read of WGBS when using certain Real Time Analysis (RTA) software versions on the HiSeq X sequencer, reminiscent of an issue that was previously reported with certain HiSeq 2500 software versions. However, with the HD.3.4.0/RTA 2.7.7 software upgrade for the HiSeq X system, we observed an overall improved quality and yield of the WGBS data generated, which in turn empowers cost-effective and high quality DNA methylation studies.

genomics↗

bioRxiv2017

De Novo Mutations Implicate Novel Genes With Burden Of Rare Variants In Systemic Lupus Erythematosus

The omnigenic model of complex diseases stipulates that the majority of the heritability will be explained by the effects of common variation on genes in the periphery of core disease pathways. Rare variant associations, expected to explain far less of the heritability, may be enriched in core disease genes and thus will be instrumental in the understanding of complex disease pathogenesis and their potential therapeutic targets. Here, using complementary whole-exome sequencing (WES), high-density imputation, and in vitro cellular assays, we identify three candidate core genes in the pathogenesis of Systemic Lupus Erythematosus (SLE). Using extreme-phenotype sampling, we sequenced the exomes of 30 SLE parent-affected-offspring trios and identified 14 genes with missense de novo mutations (DNM), none of which are within the >80 SLE susceptibility loci implicated through genome-wide association studies (GWAS). In a follow-up cohort of 10,995 individuals of matched European ancestry, we imputed genotype data to the density of the combined UK10K-1000 genomes Phase III reference panel across the 14 candidate genes. We identify a burden of rare variants across PRKCD associated with SLE risk (P=0.0028), and across DNMT3A associated with two severe disease prognosis sub-phenotypes (P=0.0005 and P=0.0033). Both genes are functional candidates and significantly constrained against missense mutations in gene-level analyses, along with C1QTNF4. We further characterise the TNF-dependent functions of candidate gene C1QTNF4 on NF-{kappa}B activation and apoptosis, which are inhibited by the p.His198Gln DNM. Our results support extreme-phenotype sampling and DNM gene discovery to aid the search for core disease genes implicated through rare variation.\n\nSignificance StatementRare variants, present in <1% in population, are expected to explain little of the heritability of complex diseases, such as Systemic Lupus Erythematosus (SLE), yet are likely to identify core genes crucial to disease mechanisms. Their rarity, however, limits the power to show their statistical association with disease. Through sequencing the exomes of SLE patients and their parents, we identified non-inherited de novo mutations in 14 genes and hypothesised that these are prime candidates for harbouring additional disease-associated rare variants. We demonstrate that two of these genes also carry a significant excess of rare variants in an independent, large cohort of SLE patients. Our findings will influence future study designs in the search for the missing heritability of complex diseases.

genetics↗