Mapping Protein-DNA Interactions Using ChIP-exo and Illumina-Based Sequencing

Chromatin immunoprecipitation (ChIP) provides a means of enriching DNA associated with transcription factors, histone modifications, and indeed any other proteins for which suitably characterized antibodies are available. Over the years, sequence detection has progressed from quantitative real-time PCR and Southern blotting to microarrays (ChIP-chip) and now high-throughput sequencing (ChIP-seq). This progression has vastly increased the sequence coverage and data volumes generated. This in turn has enabled informaticians to predict the identity of multi-protein complexes on DNA based on the overrepresentation of sequence motifs in DNA enriched by ChIP with a single antibody against a single protein. In the course of the development of high-throughput sequencing, little has changed in the ChIP methodology until recently. In the last three years, a number of modifications have been made to the ChIP protocol with the goal of enhancing the sensitivity of the method and further reducing the levels of nonspecific background sequences in ChIPped samples. In this chapter, we provide a brief commentary on these methodological changes and describe a detailed ChIP-exo method able to generate narrower peaks and greater peak coverage from ChIPped material.

Inter-Laboratory Evaluation of a Next-Generation Sequencing Panel for Acute Myeloid Leukemia

INTRODUCTION: Acute myeloid leukemia (AML) is a heterogeneous clonal disorder often associated with dismal overall survival. The clinical diversity of AML is reflected in the range of recurrent somatic mutations in several genes, many of which have a prognostic and therapeutic value. Targeted next-generation sequencing (NGS) of these genes has the potential for translation into clinical practice. In order to assess this potential, an inter-laboratory evaluation of a commercially available AML gene panel across three diagnostic centres in the UK and Ireland was performed.

METHODS: DNA from six AML patient samples was distributed to each centre and processed using a standardised workflow, including a common sequencing platform, sequencing chips and bioinformatics pipeline. A duplicate sample in each centre was run to assess inter- and intra-laboratory performance.

RESULTS: An average sample read depth of 2725X (range 629-5600) was achieved using six samples per chip, with some variability observed in the depth of coverage generated for individual samples and between centres. A total of 16 somatic mutations were detected in the six AML samples, with a mean of 2.7 mutations per sample (range 1-4) representing nine genes on the panel. 15/16 mutations were identified by all three centres. Allelic frequencies of the mutations ranged from 5.6 to 53.3 % (median 44.4 %), with a high level of concordance of these frequencies between centres, for mutations detected.

CONCLUSION: In this inter-laboratory comparison, a high concordance, reproducibility and robustness was demonstrated using a commercially available NGS AML gene panel and platform.

Optimisation of a droplet digital PCR assay for the diagnosis of Schistosoma japonicum infection: A duplex approach with DNA binding dye chemistry

Schistosomiasis is a chronically debilitating helminth infection with a significant socio-economic and public health impact. Accurate diagnostics play a pivotal role in achieving current schistosomiasis control and elimination goals. However, many of the current diagnostic procedures, which rely on detection of schistosome eggs, have major limitations including lack of accuracy and the inability to detect pre-patent infections. DNA-based detection methods provide a viable alternative to the current tests commonly used for schistosomiasis diagnosis. Here we describe the optimisation of a novel droplet digital PCR (ddPCR) duplex assay for the diagnosis of Schistosoma japonicum infection which provides improved detection sensitivity and specificity. The assay involves the amplification of two specific and abundant target gene sequences in S. japonicum; a retrotransposon (SjR2) and a portion of a mitochondrial gene (nad1). The assay detected target sequences in different sources of schistosome DNA isolated from adult worms, schistosomules and eggs, and exhibits a high level of specificity, thereby representing an ideal tool for the detection of low levels of parasite DNA in different clinical samples including parasite cell free DNA in the host circulation and other bodily fluids. Moreover, being quantitative, the assay can be used to determine parasite infection intensity and, could provide an important tool for the detection of low intensity infections in low prevalence schistosomiasis-endemic areas.

Understanding next generation sequencing in oncology: A guide for oncologists

DNA sequencing is now faster and cheaper than ever before, due to the development of next generation sequencing (NGS) technologies. NGS is now widely used in the research setting and is becoming increasingly utilised in clinical practice. However, due to evolving clinical commitments, increased workload and lack of training opportunities, many oncologists may be unfamiliar with the terminology and technology involved. This can lead to oncologists feeling daunted by issues such as how to interpret the vast amounts of data generated by NGS and the differences between sequencing platforms. This review article explains common concepts and terminology, summarises the process of DNA sequencing (including data analysis) and discusses the main factors to consider when deciding on a sequencing method. This article aims to improve oncologists' understanding of the most commonly used sequencing platforms and the ongoing challenges faced in expanding the use of NGS into routine clinical practice.

Genetics and Prognostication in Splenic Marginal Zone Lymphoma: Revelations from Deep Sequencing

Purpose: Mounting evidence supports the clinical significance of gene mutations and immunogenetic features in common mature B-cell malignancies.

Experimental Design: We undertook a detailed characterization of the genetic background of splenic marginal zone lymphoma (SMZL), using targeted resequencing and explored potential clinical implications in a multinational cohort of 175 patients with SMZL.

Results: We identified recurrent mutations in TP53 (16%), KLF2 (12%), NOTCH2 (10%), TNFAIP3 (7%), MLL2 (11%), MYD88 (7%), and ARID1A (6%), all genes known to be targeted by somatic mutation in SMZL. KLF2 mutations were early, clonal events, enriched in patients with del(7q) and IGHV1-2*04 B-cell receptor immunoglobulins, and were associated with a short median time to first treatment (0.12 vs. 1.11 years; P = 0.01). In multivariate analysis, mutations in NOTCH2 [HR, 2.12; 95% confidence interval (CI), 1.02–4.4; P = 0.044] and 100% germline IGHV gene identity (HR, 2.19; 95% CI, 1.05–4.55; P = 0.036) were independent markers of short time to first treatment, whereas TP53 mutations were an independent marker of short overall survival (HR, 2.36; 95 % CI, 1.08–5.2; P = 0.03).

Conclusions: We identify key associations between gene mutations and clinical outcome, demonstrating for the first time that NOTCH2 and TP53 gene mutations are independent markers of reduced treatment-free and overall survival, respectively.

Full-Duplex Spectrum Sharing in Cooperative Single Carrier Systems

We propose cyclic prefix single carrier full-duplex transmission in amplify-and-forward cooperative spectrum sharing networks to achieve multipath diversity and full-duplex spectral efficiency. Integrating full-duplex transmission into cooperative spectrum sharing systems results in two intrinsic problems: 1) the residual loop interference occurs between the transmit and the receive antennas at the secondary relays and 2) the primary users simultaneously suffer interference from the secondary source (SS) and the secondary relays (SRs). Thus, examining the effects of residual loop interference under peak interference power constraint at the primary users and maximum transmit power constraints at the SS and the SRs is a particularly challenging problem in frequency selective fading channels. To do so, we derive and quantitatively compare the lower bounds on the outage probability and the corresponding asymptotic outage probability for max–min relay selection, partial relay selection, and maximum interference relay selection policies in frequency selective fading channels. To facilitate comparison, we provide the corresponding analysis for half-duplex. Our results show two complementary regions, named as the signal-to-noise ratio (SNR) dominant region and the residual loop interference dominant region, where the multipath diversity and spatial diversity can be achievable only in the SNR dominant region, however the diversity gain collapses to zero in the residual loop interference dominant region.

Secure Full-Duplex Small-Cell Networks in a Spectrum Sharing Environment

In this paper, we propose three relay selection schemes for full-duplex heterogeneous networks in the presence of multiple cognitive radio eavesdroppers. In this setup, the cognitive small-cell nodes (secondary network) can share the spectrum licensed to the macro-cell system (primary network) on the condition that the quality-of-service of the primary network is always satisfied subjected to its outage probability constraint. The messages are delivered from one small-cell base station to the destination with the help of full-duplex small-cell base stations, which act as relay nodes. Based on the availability of the network’s channel state information at the secondary information source, three different selection criteria for full-duplex relays, namely: 1) partial relay selection; 2) optimal relay selection; and 3) minimal self-interference relay selection, are proposed. We derive the exact closed-form and asymptotic expressions of the secrecy outage probability for the three criteria under the attack of non-colluding/colluding eavesdroppers. We demonstrate that the optimal relay selection scheme outperforms the partial relay selection and minimal self-interference relay selection schemes at the expense of acquiring full channel state information knowledge. In addition, increasing the number of the full-duplex small-cell base stations can improve the security performance. At the illegitimate side, deploying colluding eavesdroppers and increasing the number of eavesdroppers put the confidential information at a greater risk. Besides, the transmit power and the desire outage probability of the primary network have great influences on the secrecy outage probability of the secondary network. 

Multipair massive MIMO full-duplex relaying with MRC/MRT processing

We consider a multipair relay channel, where multiple sources communicate with multiple destinations with the help of a full-duplex (FD) relay station (RS). All sources and destinations have a single antenna, while the RS is equipped with massive arrays. We assume that the RS estimates the channels by using training sequences transmitted from sources and destinations. Then, it uses maximum-ratio combining/maximum-ratio transmission (MRC/MRT) to process the signals. To significantly reduce the loop interference (LI) effect, we propose two massive MIMO processing techniques: i) using a massive receive antenna array; or ii) using a massive transmit antenna array together with very low transmit power at the RS. We derive an exact achievable rate in closed-form and evaluate the system spectral efficiency. We show that, by doubling the number of antennas at the RS, the transmit power of each source and of the RS can be reduced by 1.5 dB if the pilot power is equal to the signal power and by 3 dB if the pilot power is kept fixed, while maintaining a given quality-of-service. Furthermore, we compare FD and half-duplex (HD) modes and show that FD improves significantly the performance when the LI level is low.