Short single-stranded DNAs with putative non-canonical structures comprise a new class of plasma cell-free DNA

Background Cell-free DNA (cfDNA), which is extracellular DNA present in the circulating plasma and other body fluids, is currently investigated as a minimally invasive, highly informative biomarker. While nucleosome-sized cfDNA fragments have been investigated intensively, shorter DNA fragments in the plasma have not been studied due to several technical limitations. Results We aimed to investigate the existence of shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in length in the human plasma, present at a molar concentration comparable to that of nucleosome-sized fragments. Unfortunately, these short fragments cannot be recovered by widely used cfDNA isolation methods. In addition, they are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. Therefore, we established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of the peaks by these short fragments. Conclusions We propose a new class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

10x Single Cell Multiome Nuclei Isolation and Library Preparation Protocol v1

10x single-cell multiome nuclei isolation and library preparation protocol for HuBMAP colon project.

GT-seq Library Preparation Protocol v1

Protocol for Genotyping-in-Thousands by sequencing (GT-seq) library preparation.

Mini-XT, a miniaturized tagmentation-based protocol for efficient sequencing of SARS-CoV-2

Introduction: The COVID-19 pandemic has highlighted the importance of whole genome sequencing (WGS) of SARS-CoV-2 to inform public health policy. By enabling definition of lineages it facilitates tracking of the global spread of the virus. The evolution of new variants can be monitored and knowledge of specific mutations provides insights into the mechanisms through which the virus increases transmissibility or evades immunity. To date almost one million SARS-CoV-2 genomes have been sequenced by members of the COVID-19 Genomics UK (COG-UK) Consortium. To achieve similar feats in a more cost-effective and sustainable manner in future, improved high throughput virus sequencing protocols are required. We have therefore developed a miniaturized library preparation protocol with drastically reduced consumable use and costs. Methods: SARS-CoV-2 RNA was amplified using the ARTIC nCov-2019 multiplex RT-PCR protocol and purified using a conventional liquid handling system. Acoustic liquid transfer (Echo 525) was employed to reduce reaction volumes and the number of tips required for a Nextera XT library preparation. Sequencing was performed on an Illumina MiSeq. Results: We present the 'Mini-XT' miniaturized tagmentation-based library preparation protocol available on protocols.io (https://dx.doi.org/10.17504/protocols.io.bvntn5en). The final version of Mini-XT has been used to sequence 4,384 SARS-CoV-2 samples from N. Ireland with a COG-UK QC pass rate of 97.4%. Sequencing quality was comparable and lineage calling consistent for replicate samples processed with full volume Nextera DNA Flex (333 samples) or using nanopore technology (20 samples). SNP calling between Mini-XT and these technologies was consistent and sequences from replicate samples paired together in maximum likelihood phylogenetic trees. Conclusion: The Mini-XT protocol maintains sequence quality while reducing library preparation reagent volumes 8-fold and halving overall tip usage from sample to sequence to provide concomitant cost savings relative to standard protocols. This will enable more efficient high-throughput sequencing of SARS-CoV-2 isolates and future pathogen WGS.

SARS-CoV-2 DNA library preparation using an adapted version of Illumina DNA prep protocol v.1.1 v1

This protocol describes the steps to prepare DNA libraries from PCR amplicons using the Illumina DNA prep library kit. The current library preparation protocol was adapted from the original Illumina DNA Prep Reference Guide (document #1000000025416 v09) using low input DNA samples as the starting material. We omitted laboratory equipment such as the Eppendorf 96-well PCR plate, microseal adhesive seals. In addition, we replaced 96-well plate magnetic stand with a magnetic stand suitable for 1.5 ml tubes. The added value of this protocol is that PCR reactions happen in 0.2 ml PCR tubes, and it can be implemented without a separate purchase of 96-well PCR plates or a magnetic stand for PCR plates. Using individual 0.2 ml tubes increases the user flexibility when running few samples for library preparation. The other advantage of using our adapted protocol is the reduced library preparation cost when running few specimens. For instance, implementing the current protocol might be cheaper than the original Illumina protocol when using the Illumina® DNA Prep, (M) Tagmentation (24 Samples) catalog number 20018704. The protocol has proven effective for processing hundreds of DNA libraries from tiled virus amplicons such as Sapovirus and SARS-CoV-2 submitted to public repositories such as GISAID and GenBank.

MiniXT protocol v1

We present the ‘mini-XT’ miniaturized tagmentation-based library preparation protocol used for Illumina WGS of SARS-CoV-2 positive samples. Reverse transcription and amplification is based upon the nCoV-2019 sequencing protocol v3 (LoCost)V.3 by Josh Quick. The key new feature of the protocol is the use of acoustic liquid transfer to automate and reduce volumes during library preparation. It is optimized for the sequencing of 384 samples, offering reduced consumable use and costs and improved throughput.

Short single-stranded DNA with putative non-canonical structures comprises a novel class of plasma cell-free DNA

Cell-free DNA (cfDNA) in human blood is currently investigated as a minimally invasive, highly informative biomarker. Here, we aimed to investigate the existence of the shorter cfDNA fragments in the blood. Using an improved cfDNA purification protocol and a 3′-end-labeling method, we found DNA fragments of approximately 50 nucleotides in human plasma, present at a molar concentration comparable to that of the nucleosome-sized fragments. These short fragments cannot be recovered by widely used cfDNA isolation methods, and are composed of single-stranded DNA (ssDNA), thus escaping detection in previous studies. We established a library-preparation protocol based on our unique ssDNA ligation technique and applied it to the isolated cfDNA. Deep sequencing of these libraries revealed that the short fragments are derived from hundreds of thousands of genomic sites in open chromatin regions and enriched with transcription factor-binding sites. Remarkably, antisense strands of putative G-quadruplex motifs occupy as much as one-third of peaks called with these short fragments. Hence, we propose a novel class of plasma cfDNA composed of short single-stranded fragments that potentially form non-canonical DNA structures.

Application of transposon-insertion sequencing to determine gene essentiality in the acetogen Clostridium autoethanogenum

The majority of the genes present in bacterial genomes remain poorly characterised with up to one third of those that are protein encoding having no definitive function. Transposon insertion sequencing represents a high-throughput technique that can help rectify this deficiency. The technology, however, can only be realistically applied to easily transformable species leaving those with low DNA-transfer rates out of reach. Here we have developed a number of approaches that overcome this barrier in the autotrophic species Clostridium autoethanogenum using a mariner-based transposon system. The inherent instability of such systems in the Escherichia coli conjugation donor due to transposition events was counteracted through the incorporation of a conditionally lethal codA marker on the plasmid backbone. Relatively low frequencies of transformation of the plasmid into C. autoethanogenum were circumvented through the use of a plasmid that is conditional for replication coupled with the routine implementation of an Illumina library preparation protocol that eliminates plasmid-based reads. A transposon library was then used to determine the essential genes needed for growth using carbon monoxide as a sole carbon and energy source.