Three Reagents for in-Solution Enrichment of Ancient Human DNA at More than a Million SNPs

In-solution enrichment for hundreds of thousands of single nucleotide polymorphisms (SNPs) has been the source of >70% of all genome-scale ancient human DNA data published to date. This approach has made it possible to generate data for one to two orders of magnitude lower cost than random shotgun sequencing, making it economical to study ancient samples with low proportions of human DNA, and increasing the rate of conversion of sampled remains into working data thereby facilitating ethical stewardship of human remains. So far, nearly all ancient DNA data obtained using in-solution enrichment has been generated using a set of bait sequences targeting about 1.24 million SNPs (the 1240k reagent). These sequences were published in 2015, but synthesis of the reagent has been cost-effective for only a few laboratories. In 2021, two companies made available reagents that target the same core set of SNPs along with supplementary content. Here, we test the properties of the three reagents on a common set of 27 ancient DNA libraries across a range of richness of DNA content and percentages of human molecules. All three reagents are highly effective at enriching many hundreds of thousands of SNPs. For all three reagents and a wide range of conditions, one round of enrichment produces data that is as useful as two rounds when tens of millions of sequences are read out as is typical for such experiments. In our testing, the Twist Ancient DNA reagent produces the highest coverages, greatest uniformity on targeted positions, and almost no bias toward enriching one allele more than another relative to shotgun sequencing. Allelic bias in 1240k enrichment has made it challenging to carry out joint analysis of these data with shotgun data, creating a situation where the ancient DNA community has been publishing two important bodes of data that cannot easily be co-analyzed by population genetic methods. To address this challenge, we introduce a subset of hundreds of thousands of SNPs for which 1240k data can be effectively co-analyzed with all other major data types.

Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed. This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e

LuxRep: a technical replicate-aware method for bisulfite sequencing data analysis

Background DNA methylation is commonly measured using bisulfite sequencing (BS-seq). The quality of a BS-seq library is measured by its bisulfite conversion efficiency. Libraries with low conversion rates are typically excluded from analysis resulting in reduced coverage and increased costs. Results We have developed a probabilistic method and software, LuxRep, that implements a general linear model and simultaneously accounts for technical replicates (libraries from the same biological sample) from different bisulfite-converted DNA libraries. Using simulations and actual DNA methylation data, we show that including technical replicates with low bisulfite conversion rates generates more accurate estimates of methylation levels and differentially methylated sites. Moreover, using variational inference speeds up computation time necessary for whole genome analysis. Conclusions In this work we show that taking into account technical replicates (i.e. libraries) of BS-seq data of varying bisulfite conversion rates, with their corresponding experimental parameters, improves methylation level estimation and differential methylation detection.

Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed.

Construction of Aerobic/Anaerobic-Substrate-Induced Gene Expression Procedure for Exploration of Metagenomes From Subseafloor Sediments

Substrate-induced gene expression (SIGEX) is a high-throughput promoter-trap method. It is a function-based metagenomic screening tool that relies on transcriptional activation of a reporter gene green fluorescence protein (gfp) by a metagenomic DNA library upon induction with a substrate. However, its use is limited because of the relatively small size of metagenomic DNA libraries and incompatibility with screening metagenomes from anaerobic environments. In this study, these limitations of SIGEX were addressed by fine-tuning metagenome DNA library construction protocol and by using Evoglow, a green fluorescent protein that forms a chromophore even under anaerobic conditions. Two metagenomic libraries were constructed for subseafloor sediments offshore Shimokita Peninsula (Pacific Ocean) and offshore Joetsu (Japan Sea). The library construction protocol was improved by (a) eliminating short DNA fragments, (b) applying topoisomerase-based high-efficiency ligation, (c) optimizing insert DNA concentration, and (d) column-based DNA enrichment. This led to a successful construction of metagenome DNA libraries of approximately 6 Gbp for both samples. SIGEX screening using five aromatic compounds (benzoate, 3-chlorobenzoate, 3-hydroxybenzoate, phenol, and 2,4-dichlorophenol) under aerobic and anaerobic conditions revealed significant differences in the inducible clone ratios under these conditions. 3-Chlorobenzoate and 2,4-dichlorophenol led to a higher induction ratio than that for the other non-chlorinated aromatic compounds under both aerobic and anaerobic conditions. After the further screening of induced clones, a clone induced by 3-chlorobenzoate only under anaerobic conditions was isolated and characterized. The clone harbors a DNA insert that encodes putative open reading frames of unknown function. Previous aerobic SIGEX attempts succeeded in the isolation of gene fragments from anaerobes. This study demonstrated that some gene fragments require a strict in vivo reducing environment to function and may be potentially missed when screened by aerobic induction. The newly developed anaerobic SIGEX scheme will facilitate functional exploration of metagenomes from the anaerobic biosphere.

OverFlap PCR – a reliable approach for generation of plasmid DNA libraries containing random sequences without template bias.

Over the decades the improvement of naturally occurring proteins and creation of novel ones has been the primary goal for many practical biotechnology researchers and it is widely recognized that randomization of protein sequences coupled to various effect screening methodologies is one of the most powerful techniques for fast, efficient and purposeful approach for acquisition of desired improvements. Over the years considerable advancements have been made in this field, however development of PCR based or template guided methodologies has been hampered by the resulting template sequence bias. In this article we present novel whole plasmid amplification based approach, which we named OverFlap PCR, for randomization of virtually any region of the plasmid DNA, without introduction of mentioned bias.

Diagnostics of hereditary cancer syndromes by NGS. Process of creating a database.

Background:more than 500 thousand new cases of malignant neoplasms are registered annually in the Russian Federation, of which more than 50 thousand new cases are due to hereditary forms.Improving the diagnosis of these diseases will make it possible to detect tumors in the early stages and take timely preventive and therapeutic measures. Aims:creation of a database and development of software for NGS data analysis for the prevention and early diagnosis of hereditary forms of oncological diseases. Methods:the present study used 636 DNA samples obtained from cancer patients with a high hereditary risk or a burdened family history. DNA was isolated from blood lymphocytes. DNA libraries were prepared with a KAPA Target Enrichment Custom Panel (Roche). The panel included probes for targeted enrichment of the coding region of 44 genes. NGS was performed on the MiSeq platform (Illumina). Results:we identified 65 pathogenic/ probably pathogenic nucleotide sequence variants in 96 patients in theATM, BLM, BRCA1, BRCA2, CHEK2, EPCAM, MEN1, MLH1, MSH2, MSH3, MSH6, MUTYH, PALB2, TP53genes. We also identified 2858 nucleotide sequence variants of unknown clinical significance. Conclusions:we have created a local database that contains both genetic variants and clinical and anamnestic data. The database contains 4763 nucleotide sequence variants at the moment, among which 2522 are unique variants identified in a single patient.

Spacer2PAM: A computational framework for identification of functional PAM sequences for endogenous CRISPR systems

RNA-guided nucleases from clustered regularly interspaced short palindromic repeats (CRISPR) systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR systems in many bacteria and archaea are particularly attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR effectors. However, each CRISPR system recognizes a unique set of PAM sequences, which requires extensive screening of randomized DNA libraries. This challenge makes it difficult to develop endogenous CRISPR systems, especially in organisms that are slow-growing or have transformation idiosyncrasies. To address this limitation, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to identify potential PAM sequences for any CRISPR system given its corresponding CRISPR array as input. Spacer2PAM can be used in Quick mode to generate a single PAM prediction that is likely to be functional or in Comprehensive mode to inform targeted, unpooled PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-predicted PAM library for the type I-B CRISPR system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR systems for industrial biotechnology and synthetic biology.

Third Generation Cytogenetic Analysis (TGCA): diagnostic application of long-read sequencing.

Unbalanced Structural Variants (uSVs) play important roles in the pathogenesis of several genetic syn- dromes. Traditional and molecular karyotyping are considered the first-tier diagnostic tests to detect macroscopic and cryptic deletions/duplications. However, their time-consuming and laborious experi- mental protocols protract diagnostic times from three to fifteen days. Long read sequencing approaches, such as Oxford Nanopore Technologies (ONT), have the ability to reduce time to results for the detection of uSVs with the same resolution of current state-of-the-art diagnostic tests. Here we compared ONT to molecular karyotyping for the detection of pathogenic uSVs of 7 patients with previously diagnosed causative CNVs of different sizes and allelic fractions. Larger chromosomal anomalies included trisomy 21 and mosaic tetrasomy 12p. Among smaller CNVs we tested two recip- rocal genomic imbalances in 7q11.23 (1.367 Mb), a 170 kb deletion encompassing NRXN1 and mosaic 6q27 (1.231 Mb) and 2q23.1 (408 kb) deletions. DNA libraries were prepared following ONT standard protocols and sequenced on the GridION device for 48 h. Data generated during runs were analysed in online mode, using NanoGLADIATOR. We were capable to identify all pathogenic CNVs with detection time inversely proportional to size and allelic fraction. Aneuploidies were called after only 30 minutes of sequencing, while 30 hours were needed to call CNVs < 500 kb also in mosaic state (44%). These results demonstrate the clinical utility of our approach that allows the molecular diagnosis of genomic disorders within a 30 minutes to 30 hours time-frame.