Comparative Performance of the MGISEQ-2000 and Illumina X-Ten Sequencing Platforms for Paleogenomics

The MGISEQ-2000 sequencer is widely used in various omics studies, but the performance of this platform for paleogenomics has not been evaluated. We here compare the performance of MGISEQ-2000 with the Illumina X-Ten on ancient human DNA using four samples from 1750BCE to 60CE. We found there were only slight differences between the two platforms in most parameters (duplication rate, sequencing bias, θ, δS, and λ). MGISEQ-2000 performed well on endogenous rate and library complexity although X-Ten had a higher average base quality and lower error rate. Our results suggest that MGISEQ-2000 and X-Ten have comparable performance, and MGISEQ-2000 can be an alternative platform for paleogenomics sequencing.

Prime-seq, efficient and powerful bulk RNA-sequencing

With the advent of Next Generation Sequencing, RNA-sequencing (RNA-seq) has become the major method for quantitative gene expression analysis. Reducing library costs by early barcoding has propelled single-cell RNA-seq, but has not yet caught on for bulk RNA-seq. Here, we optimized and validated a bulk RNA-seq method we call prime-seq. We show that with respect to library complexity, measurement accuracy, and statistical power it performs equivalent to TruSeq, a standard bulk RNA-seq method, but is four-fold more cost-efficient due to almost 50-fold cheaper library costs. We also validate a direct RNA isolation step that further improves cost and time-efficiency, show that intronic reads are derived from RNA, validate that prime-seq performs optimal with only 1,000 cells as input, and calculate that prime-seq is the most cost-efficient bulk RNA-seq method currently available. We discuss why many labs would profit from a cost-efficient early barcoding RNA-seq protocol and argue that prime-seq is well suited for setting up such a protocol as it is well validated, well documented, and requires no specialized equipment.

PEPPRO: quality control and processing of nascent RNA profiling data

Nascent RNA profiling is growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable workflow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniformly processed output files for downstream analysis and assesses adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report. PEPPRO can be run locally or using a cluster, providing a portable first step for genomic nascent RNA analysis.

Transposase-assisted tagmentation of RNA/DNA hybrid duplexes

Tn5-mediated transposition of double-strand DNA has been widely utilized in various high-throughput sequencing applications. Here, we report that the Tn5 transposase is also capable of direct tagmentation of RNA/DNA hybrids in vitro. As a proof-of-concept application, we utilized this activity to replace the traditional library construction procedure of RNA sequencing, which contains many laborious and time-consuming processes. Results of Transposase-assisted RNA/DNA hybrids Co-tagmEntation (termed ‘TRACE-seq’) are compared to traditional RNA-seq methods in terms of detected gene number, gene body coverage, gene expression measurement, library complexity, and differential expression analysis. At the meantime, TRACE-seq enables a cost-effective one-tube library construction protocol and hence is more rapid (within 6 hr) and convenient. We expect this tagmentation activity on RNA/DNA hybrids to have broad potentials on RNA biology and chromatin research.

Bottom-Up Design Approach for OBOC Peptide Libraries

One-bead-one-compound peptide libraries, developed following the top-down experimental approach, have attracted great interest in the identification of potential ligands or active peptides. By exploiting a reverse experimental design approach based on the bottom-up strategy, we aimed to develop simplified, maximally diverse peptide libraries that resulted in the successful characterization of mixture components. We show that libraries of 32 and 48 components can be successfully detected in a single run using chromatography coupled to mass spectrometry (UPLC-MS). The proposed libraries were further theoretically evaluated in terms of their composition and physico-chemical properties. By combining the knowledge obtained on single libraries we can cover larger sequence spaces and provide a controlled exploration of the peptide chemical space both theoretically and experimentally. Designing libraries by using the bottom-up approach opens up the possibility of rationally fine-tuning the library complexity based on the available analytical methods.

A High-Throughput Single-Clone Phage Fluorescence Microwell Immunoassay and Laser-Driven Clonal Retrieval System

Phage display is one of the most frequently used platform technologies utilized to screen and select therapeutic antibodies, and has contributed to the development of more than 10 therapeutic antibodies used in the clinic. Despite advantages like efficiency and low cost, it has intrinsic technical limitations, such as the asymmetrical amplification of the library after each round of biopanning, which is regarded as a reason for it yielding a very limited number of antigen binders. In this study, we developed a high-throughput single-clonal screening system comprised of fluorescence immunoassays and a laser-driven clonal DNA retrieval system using microchip technology. Using this system, from a single-chain variable fragment (scFv) library displayed on phages with a complexity of 5.21 × 105 harboring random mutations at five amino acid residues, more than 70,000 clones—corresponding to ~14% of the library complexity—were screened, resulting in 78 antigen-reactive scFv sequences with mutations restricted to the randomized residues. Our results demonstrate that this system can significantly reduce the number of biopanning rounds, or even eliminate the need for this process for libraries with lower complexity, providing an opportunity to obtain more diverse clones from the library.

Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline

Methylation at the N6 position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes, however accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-Seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP) based approaches that rely on inducing specific mutations during reverse transcription via UV crosslinking of the anti-m6A antibody to methylated RNA have been employed to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.

Quality control and processing of nascent RNA profiling data

Experiments that profile nascent RNA are growing in popularity; however, there is no standard analysis pipeline to uniformly process the data and assess quality. Here, we introduce PEPPRO, a comprehensive, scalable workflow for GRO-seq, PRO-seq, and ChRO-seq data. PEPPRO produces uniform processed output files for downstream analysis, including alignment files, signal tracks, and count matrices. Furthermore, PEPPRO simplifies downstream analysis by using a standard project definition format which can be read using metadata APIs in R and Python. For quality control, PEPPRO provides several novel statistics and plots, including assessments of adapter abundance, RNA integrity, library complexity, nascent RNA purity, and run-on efficiency. PEPPRO is restartable and fault-tolerant, records copious logs, and provides a web-based project report for navigating results. It can be run on local hardware or using any cluster resource manager, using either native software or a provided modular Linux container environment. PEPPRO is thus a robust and portable first step for genomic nascent RNA analysis.AvailabilityBSD2-licensed code and documentation: https://peppro.databio.org.