SuperSelective primer pairs for sensitive detection of rare somatic mutations

SuperSelective primers, by virtue of their unique design, enable the selective exponential amplification of rare DNA fragments containing somatic mutations in the presence of abundant closely related wild-type DNA fragments. However, when a SuperSelective primer is used in conjunction with a conventional reverse primer, linear amplification of the abundant wild-type fragments occurs, and this may lead to a late arising signal that can be confused with the late arising signal from the rare mutant fragments. We have discovered that the use of a pair of SuperSelective primers, one specific for the target mutation in a plus strand, and the other specific for the same mutation in the complementary minus strand, but both possessing 3′-terminal nucleotides that are complementary to the mutation, significantly suppresses the linear amplification of the related wild-type sequence, and prevents the generation of false mutant sequences due to mis-incorporation by the DNA polymerase. As a consequence, the absence of mutant fragments in a sample does not give rise to a false-positive signal, and the presence of mutant fragments in a sample is clearly distinguishable as a true-positive signal. The use of SuperSelective primer pairs should enhance the sensitivity of multiplex PCR assays that identify and quantitate somatic mutations in liquid biopsies obtained from patients with cancer, thereby enabling the choice of a targeted therapy, the determination of its effectiveness over time, and the substitution of a more appropriate therapy as new mutations arise.

High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq)

Chromatin profiling in single cells has been extremely challenging and almost exclusively limited to histone proteins. In cases where single-cell methods have shown promise, many require highly specialized equipment or cell type–specific protocols and are relatively low throughput. Here, we combine the advantages of tagmentation, linear amplification, and combinatorial indexing to produce a high-throughput single-cell DNA binding site mapping method that is simple, inexpensive, and capable of multiplexing several independent samples per experiment. Targeted insertion of promoters sequencing (TIP-seq) uses Tn5 fused to proteinA to insert a T7 RNA polymerase promoter adjacent to a chromatin protein of interest. Linear amplification of flanking DNA with T7 polymerase before sequencing library preparation provides ∼10-fold higher unique reads per single cell compared with other methods. We applied TIP-seq to map histone modifications, RNA polymerase II (RNAPII), and transcription factor CTCF binding sites in single human and mouse cells.

Testing Nonlinear Amplification Factors of Ground-Motion Models

ABSTRACT We explore nonlinear site effects in the new Japanese ground-motion dataset compiled by Bahrampouri et al. (2020). Following the approach of Seyhan and Stewart (2014), we evaluate the decrease of soil amplification according to the increasing and corresponding ground motion on surface rock (VS30=760 m/s). To better predict the rock ground motion associated with each record, we take into account the between-event variability of the ground motion, and to better evaluate the impact of nonlinearity, we correct observed ground motion on soil by the site-specific linear amplification. Instead of grouping the stations by site-response proxy, we focus on individual stations with several strong-motion records. We develop a framework to test recently published nonlinear site amplification models against a linear site amplification model and compare the results with recent building codes that include nonlinearity. The results show that the site response varies greatly from site to site, indicating that conventional site proxies, such as VS30, are not sufficient to characterize nonlinear site response. Out of all of the Kiban–Kyoshin network stations, 20 stations are selected as having recorded sufficient data to be used in the test. Out of these 20 stations, five stations show signs of nonlinearity, that is, the nonlinear models performed better than the linear-amplification model for all periods T. For most sites, however, the linear site amplification models get the best score. This suggest that, for the range of predicted rock motion considered in this study (peak ground acceleration <0.2g), nonlinearity may not have a sufficiently large impact on soil ground motion to justify the use of nonlinear site terms in ground-motion functional forms and seismic building codes for such moderate-level shaking.

Profiling the binding sites of RNA-binding protein by LACE-seq

RNA-binding proteins (RBPs) directly interact with various RNAs in living cells to regulate their processing, translation, and stability. Identifying the precise binding sites of RBPs is critical for appreciating their physiological or pathological roles in germline and early embryo development. Current methods typically need millions of cells to map RBP binding positions, which prevents us from appreciating the crucial role of RBPs in early development. Here, we present the LACE-seq method for unbiased mapping of RBP-binding sites at single-nucleotide resolution in fewer cells or even single oocytes. LACE-seq depends on RBP-mediated reverse transcription termination, and linear amplification of the cDNA ends for deep sequencing. To further promote its application, we describe a step-by-step protocol about how to construct a successful LACE-seq library.

Single-cell sequencing of the small and AT-skewed genome of malaria parasites

Single-cell genomics is a rapidly advancing field; however, most techniques are designed for mammalian cells. We present a single-cell sequencing pipeline for an intracellular parasite, Plasmodium falciparum, with a small genome of extreme base content. Through optimization of a quasi-linear amplification method, we target the parasite genome over contaminants and generate coverage levels allowing detection of minor genetic variants. This work, as well as efforts that build on these findings, will enable detection of parasite heterogeneity contributing to P. falciparum adaptation. Furthermore, this study provides a framework for optimizing single-cell amplification and variant analysis in challenging genomes.

High throughput genome-wide single cell protein:DNA binding site mapping by targeted insertion of promoters (TIP-seq)

ABSTRACTAssessing cell to cell, and importantly, chromosome to chromosome, heterogeneity in cellular phenotypes is a central goal of modern cell biology. However, chromatin profiling in single cells has been extremely challenging, and single chromosome profiling has not been achieved. In cases where single cell methods have shown promise, success has been mainly limited to histone proteins and/or require highly specialized equipment or cell type specific protocols and are relatively low throughput. Here, we have combined the advantages of tagmentation, linear amplification and combinatorial indexing to produce a high throughput single cell DNA binding site mapping method that does not require specialized equipment and is capable of multiplexing several samples/ target proteins in one experiment. Targeted Insertion of Promoters (TIP-seq) uses Tn5 fused to protein A (as with CUT&Tag) to insert a T7 RNA polymerase promoter into sites adjacent to an antibody bound to a chromatin protein of interest, followed by linear amplification of flanking DNA with T7 polymerase, cDNA preparation and PCR indexing. Tip-seq provides ∼10-fold higher unique reads and thus higher coverage per single cell compared to state-of-the-art methods. We apply TIP-seq to map histone modifications, RNA PolII and CTCF binding sites in single human and mouse cells. TIP-seq will also be adaptable for other platforms, such as 10X genomics and ICELL8. In summary, TIP-seq provides a high-throughput, low-cost method for single cell protein mapping, that yields substantially higher coverage per cell and signal to noise than existing methods.

Testing non-linear amplification factors used in ground motion models

<p>Non-linear site effects are mainly expected for strong ground motions and sites with soft soils and more recent ground-motion models (GMM) have started to include such effects. Observations in this range are, however, sparse, and most non-linear site amplification models are therefore partly or fully based on numerical simulations. We develop a framework for testing of non-linear site amplification models using data from the comprehensive Kiban-Kyoshin network in Japan. The test is reproducible, following the vision of the Collaboratory for the Study of Earthquake Predictability (CSEP), and takes advantage of new large datasets to evaluate <span>whether or not</span> non-linear site effects predicted by site-amplification models are supported by empirical data. The site amplification models are tested using residuals between the observations and predictions from a GMM based only on magnitude and distance. When the GMM is derived without any site term, the site-specific variability extracted from the residuals is expected to capture the site response of a site. The non-linear site amplification models are tested against a linear amplification model on individual well-record<span>ing</span> stations. Finally, the result is compared to building codes where non-linearity is included. The test shows that for most of the sites selected as having sufficient records, the non-linear site-amplification models do not score better than the linear amplification model. This suggests that including non-linear site amplification in GMMs and building codes may not yet be justified, at least not in the range of ground motions considered in the test (peak ground acceleration < 0.2 g).</p>

Transient Noise Reduction Using a Deep Recurrent Neural Network: Effects on Subjective Speech Intelligibility and Listening Comfort

A deep recurrent neural network (RNN) for reducing transient sounds was developed and its effects on subjective speech intelligibility and listening comfort were investigated. The RNN was trained using sentences spoken with different accents and corrupted by transient sounds, using the clean speech as the target. It was tested using sentences spoken by unseen talkers and corrupted by unseen transient sounds. A paired-comparison procedure was used to compare all possible combinations of three conditions for subjective speech intelligibility and listening comfort for two relative levels of the transients. The conditions were: no processing (NP); processing using the RNN; and processing using a multi-channel transient reduction method (MCTR). Ten participants with normal hearing and ten with mild-to-moderate hearing loss participated. For the latter, frequency-dependent linear amplification was applied to all stimuli to compensate for individual audibility losses. For the normal-hearing participants, processing using the RNN was significantly preferred over that for NP for subjective intelligibility and comfort, processing using the RNN was significantly preferred over that for MCTR for subjective intelligibility, and processing using the MCTR was significantly preferred over that for NP for comfort for the higher transient level only. For the hearing-impaired participants, processing using the RNN was significantly preferred over that for NP for both subjective intelligibility and comfort, processing using the RNN was significantly preferred over that for MCTR for comfort, and processing using the MCTR was significantly preferred over that for NP for comfort.

A homology independent sequence replacement strategy in human cells using a CRISPR nuclease

Precision genomic alterations largely rely on homology directed repair (HDR), but targeting without homology using the non-homologous end-joining (NHEJ) pathway has gained attention as a promising alternative. Previous studies demonstrated precise insertions formed by the ligation of donor DNA into a targeted genomic double-strand break in both dividing and non-dividing cells. Here, we demonstrate the use of NHEJ repair to replace genomic segments with donor sequences; we name this method ‘Replace’ editing ( R ational e nd-joining p rotocol de l ivering a targeted sequen c e e xchange). Using CRISPR/Cas9, we create two genomic breaks and ligate a donor sequence in-between. This exchange of a genomic for a donor sequence uses neither microhomology nor homology arms. We target four loci in cell lines and show successful exchange of exons in 16–54% of human cells. Using linear amplification methods and deep sequencing, we quantify the diversity of outcomes following Replace editing and profile the ligated interfaces. The ability to replace exons or other genomic sequences in cells not efficiently modified by HDR holds promise for both basic research and medicine.