• antimicrobial resistance
• bacterial genetics
• systems analysis
• dna damage and repair
• transcription

• Anti-Bacterial Agents/pharmacology
• DNA-Directed RNA Polymerases/metabolism
• DNA-Directed RNA Polymerases/genetics
• Genomic Instability
• Ribosomes/metabolism
• Ribosomes/drug effects
• Gentamicins/pharmacology
• Drug Resistance, Bacterial/genetics
• Escherichia coli/genetics
• Escherichia coli/drug effects
• Escherichia coli/metabolism
• Mutation
• Mutagenesis
• Transcription, Genetic/drug effects
• Protein Biosynthesis/drug effects

• 32170099 National Natural Science Foundation of China (National Science Foundation of China)
• 32100071 National Natural Science Foundation of China (National Science Foundation of China)
• National Key R&D Program of China (2023YFC2306300); Tsinghua University Initiative Scientific Research Program (20221080020, 20231080040).
• Chinese Postdoctoral Science Foundation (BX20190176 and 2020M670288).

• DNA damage response
• alternative lengthening of telomeres
• cancer
• telomerase inhibitor
• telomere

• R01 AI164530 NIAID NIH HHS
• R01 HG011676 NHGRI NIH HHS

RNA G-quadruplexes (rG4s) are non-canonical structural motifs that have diverse functional and regulatory roles, for instance in transcription termination, alternative splicing, mRNA localization and stabilization, and translational process. We recently developed the RNA G-quadruplex structure sequencing (rG4-seq) technique and described rG4s in both eukaryotic and prokaryotic transcriptomes. However, rG4-seq suffers from a complicated gel purification step and limited PCR product yield, thus requiring a high amount of RNA input, which limits its applicability in more physiologically or clinically relevant studies often characterized by the limited availability of biological material and low RNA abundance. Here, we redesign and enhance the workflow of rG4-seq to address this issue.

We developed rG4-seq 2.0 by introducing a new ssDNA adapter containing deoxyuridine during library preparation to enhance library quality with no gel purification step, less PCR amplification cycles and higher yield of PCR products. We demonstrate that rG4-seq 2.0 produces high-quality cDNA libraries that support reliable and reproducible rG4 identification at varying RNA inputs, including RNA mounts as low as 10 ng. rG4-seq 2.0 also improved the rG4-seq calling outcome and nucleotide bias in rG4 detection persistent in rG4-seq 1.0. We further provide in vitro mapping of rG4 in the HEK293T cell line, and recommendations for assessing RNA input and sequencing depth for individual rG4 studies based on transcript abundance.

rG4-seq 2.0 can improve the identification and study of rG4s in low abundance transcripts, and our findings can provide insights to optimize cDNA library preparation in other related methods.

The online version contains supplementary material available at 10.1186/s12915-022-01448-3.

• cancer
• cancer detection
• cell-free DNA
• liquid biopsy
• next-generation sequencing

• 61971122 National Natural Science Foundation of China

• diagnostics
• miRNA
• small RNA-seq

• P303/18/21942S Czech Science Foundation
• NU21-08-00286 Czech Health Research Council

• biomarkers
• cancer diagnostics and screening
• cell-free DNA
• epigenetics
• liquid biopsy

Liquid biopsy testing utilising Next Generation Sequencing (NGS) is rapidly moving towards clinical adoption for personalised oncology. However, before NGS can fulfil its potential any novel testing approach must identify ways of reducing errors, allowing separation of true low-frequency mutations from procedural artefacts, and be designed to improve upon current technologies. Popular NGS technologies typically utilise two DNA capture approaches; PCR and ligation, which have known limitations and seem to have reached a development plateau with only small, stepwise improvements being made. To maximise the ultimate utility of liquid biopsy testing we have developed a highly versatile approach to NGS: Adaptor Template Oligo Mediated Sequencing (ATOM-Seq). ATOM-Seq's strengths and versatility avoid the major limitations of both PCR- and ligation-based approaches. This technology is ligation free, simple, efficient, flexible, and streamlined, and it offers novel advantages that make it perfectly suited for use on highly challenging clinical material. Using reference and clinical materials, we demonstrate detection of known SNVs down to allele frequencies of 0.1% using as little as 20–25 ng of cfDNA, as well as the ability to detect fusions from RNA. We illustrate ATOM-Seq’s suitability for clinical testing by showing high concordance rates between paired cfDNA and FFPE clinical samples.

The development of high-throughput sequencing (next-generation sequencing technology (NGS)) and the continuous increase in experimental throughput require the upstream sample processing steps of NGS to be as simple as possible to improve the efficiency of the entire NGS process. The transposition system has fast “cut and paste” and “copy and paste” functions, and has been innovatively applied to the NGS field. For example, the Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-Seq) uses high-throughput sequencing to detect chromatin regions accessible by Tn5 transposase. Linear Amplification via Transposon Insertion (LIANTI) uses Tn5 transposase for linear amplification, haploid typing, and structural variation detection. Not only is it efficient and simple, it effectively shortens the time for NGS sample library construction, realizes large-scale and rapid sequencing, improves sequencing resolution, and can be flexibly modified for more technological innovation.

• 3D genome structures
• Tn5
• epigenetics
• genomic variation
• long fragments
• open chromatin

• Animals
• Chromatin/genetics
• Epigenomics/methods
• Genetic Variation/genetics
• Genomics/methods
• High-Throughput Nucleotide Sequencing/methods
• Humans
• Sequence Analysis, DNA/methods
• Transposases/genetics
• Transposases/metabolism
• Transposases/physiology

Small RNAs are important regulators of gene expression and are involved in human development and disease. Next generation sequencing (NGS) allows for scalable, genome-wide studies of small RNA; however, current methods are challenged by low sensitivity and high bias, limiting their ability to capture an accurate representation of the cellular small RNA population. Several studies have shown that this bias primarily arises during the ligation of single-strand adapters during library preparation, and that this ligation bias is magnified by 2′-O-methyl modifications (2′OMe) on the 3′ terminal nucleotide. In this study, we developed a novel library preparation process using randomized splint ligation with a cleavable adapter, a design which resolves previous challenges associated with this ligation strategy. We show that a randomized splint ligation based workflow can reduce bias and increase the sensitivity of small RNA sequencing for a wide variety of small RNAs, including microRNA (miRNA) and tRNA fragments as well as 2′OMe modified RNA, including Piwi-interacting RNA and plant miRNA. Finally, we demonstrate that this workflow detects more differentially expressed miRNA between tumorous and matched normal tissues. Overall, this library preparation process allows for highly accurate small RNA sequencing and will enable studies of 2′OMe modified RNA with new levels of detail.

• ATAC-seq, Assay for Transposase-Accessible Chromatin-sequencing and high-throughput sequencing
• AUC, Area Under Curve
• Biomarkers
• CTC, circulating tumor cell
• Cell-free DNA
• ESCA, esophageal cancer
• Esophageal cancer
• NGS, next generation sequencing
• NIPT, noninvasive prenatal testing
• Next generation sequencing
• PCA, principal component analysis
• SALP-seq
• SALP-seq, Single strand Adaptor Library Preparation-sequencing
• SNP, single nucleotide polymorphism
• SNV, single nucleotide variant
• TCGA, The Cancer Genome Atlas
• TF, transcription factor
• TFBS, TF binding site
• TSS, transcription start site
• Ti, transitions
• Tv, transversion
• cfDNA, cell-free DNA
• cfMeDIP-seq, cell-free methylated DNA immunoprecipitation and high-throughput sequencing
• ctDNA, cell-free tumor DNA
• mRNA, messenger RNA
• miRNA, microRNA

Cell-free DNA (cfDNA), present in circulating blood plasma, contains information about prenatal health, organ transplant reception, and cancer presence and progression. Originally developed for the genomic analysis of highly degraded ancient DNA, single-stranded DNA (ssDNA) library preparation methods are gaining popularity in the field of cfDNA analysis due to their efficiency and ability to convert short, fragmented DNA into sequencing libraries without altering DNA ends. However, current ssDNA methods are costly and time-consuming.

Here we present an efficient ligation-based single-stranded library preparation method that is engineered to produce complex libraries in under 2.5 h from as little as 1 nanogram of input DNA without alteration to the native ends of template molecules. Our method, called Single Reaction Single-stranded LibrarY or SRSLY, ligates uniquely designed Next-Generation Sequencing (NGS) adapters in a one-step combined phosphorylation/ligation reaction that foregoes end-polishing. Using synthetic DNA oligos and cfDNA, we demonstrate the efficiency and utility of this approach and compare with existing double-stranded and single-stranded approaches for library generation. Finally, we demonstrate that cfDNA NGS data generated from SRSLY can be used to analyze DNA fragmentation patterns to deduce nucleosome positioning and transcription factor binding.

SRSLY is a versatile tool for converting short and fragmented DNA molecules, like cfDNA fragments, into sequencing libraries while retaining native lengths and ends.

• Cell-free DNA
• Next-generation sequencing
• Nucleosome positioning
• Oligos
• SRSLY
• Single-stranded library

• SALP
• cell free DNA
• chromatin state
• esophageal cancer
• next-generation sequencing