Evaluation of a parasite-density based pooled targeted amplicon deep sequencing (TADS) method for molecular surveillance of Plasmodium falciparum drug resistance genes in Haiti

Sequencing large numbers of individual samples is often needed for countrywide antimalarial drug resistance surveillance. Pooling DNA from several individual samples is an alternative cost and time saving approach for providing allele frequency (AF) estimates at a population level. Using 100 individual patient DNA samples of dried blood spots from a 2017 nationwide drug resistance surveillance study in Haiti, we compared codon coverage of drug resistance-conferring mutations in four Plasmodium falciparum genes (crt, dhps, dhfr, and mdr1), for the same deep sequenced samples run individually and pooled. Samples with similar real-time PCR cycle threshold (Ct) values (+/- 1.0 Ct value) were combined with ten samples per pool. The sequencing success for samples in pools were higher at a lower parasite density than the individual samples sequence method. The median codon coverage for drug resistance-associated mutations in all four genes were greater than 3-fold higher in the pooled samples than in individual samples. The overall codon coverage distribution for pooled samples was wider than the individual samples. The sample pools with < 40 parasites/μL blood showed more discordance in AF calls for dhfr and mdr1 between the individual and pooled samples. This discordance in AF estimation may be due to low amounts of parasite DNA, which could lead to variable PCR amplification efficiencies. Grouping samples with an estimated ≥ 40 parasites/μL blood prior to pooling and deep sequencing yielded the expected population level AF. Pooling DNA samples based on estimates of > 40 parasites/μL prior to deep sequencing can be used for rapid genotyping of a large number of samples for these four genes and possibly other drug resistant markers in population-based studies. As Haiti is a low malaria transmission country with very few mixed infections and continued chloroquine sensitivity, the pooled sequencing approach can be used for routine national molecular surveillance of resistant parasites.

Comparison of double-stranded and single-stranded cfDNA library construction methods for targeted genome and methylation sequencing

Cell-free DNA (cfDNA) profiling by deep sequencing (i.e., by next generation sequencing (NGS)) has wide applications in cancer diagnosis, prognosis, and therapy response monitoring. One key step of cfDNA deep sequencing workflow is NGS library construction, whose efficiency significantly affects the utilization efficiency of cfDNA molecules, and eventually determines effective sequencing depth and sequencing accuracy. In this study, we compared two different types of cfDNA library construction methods, namely double-stranded library (dsLib, the conventional method which captures dsDNA molecules) and single-stranded library (ssLib) preparation, which captures ssDNA molecules, for the applications of mutation detection and methylation profiling, respectively. Our results suggest that the dsLib method was suitable for mutation detection while the ssLib method proved more efficient for methylation analysis. Our findings could help researchers choose the more appropriate library construction method for corresponding downstream applications of cfDNA sequencing.

Chromatin alterations during the epididymal maturation of mouse sperm refine the paternally inherited epigenome

Background Paternal lifestyle choices and male exposure history have a critical influence on the health and fitness of the next generation. Accordingly, defining the processes of germline programming is essential to resolving how the epigenetic memory of paternal experiences transmits to their offspring. Established dogma holds that all facets of chromatin organization and histone posttranslational modification are complete before sperm exits the testes. However, recent clinical and animal studies suggest that patterns of DNA methylation change during epididymal maturation. In this study, we used complementary proteomic and deep-sequencing approaches to test the hypothesis that sperm posttranslational histone modifications change during epididymal transit. Results Using proteomic analysis to contrast immature spermatozoa and mature sperm isolated from the mouse epididymis, we find progressive changes in multiple histone posttranslational modifications, including H3K4me1, H3K27ac, H3K79me2, H3K64ac, H3K122ac, H4K16ac, H3K9me2, and H4K20me3. Interestingly, some of these changes only occurred on histone variant H3.3, and most involve chromatin modifications associated with gene enhancer activity. In contrast, the bivalent chromatin modifications, H3K4me3, and H3K27me3 remained constant. Using chromatin immunoprecipitation coupled with deep sequencing, we find that changes in histone h3, lysine 27 acetylation (H3K27ac) involve sharpening broad diffuse regions into narrow peaks centered on the promoter regions of genes driving embryonic development. Significantly, many of these regions overlap with broad domains of H3K4me3 in oocytes and ATAC-seq signatures of open chromatin identified in MII oocytes and sperm. In contrast, histone h3, lysine 9 dimethylation (H3K9me2) becomes enriched within the promoters of genes driving meiosis and in the distal enhancer regions of tissue-specific genes sequestered at the nuclear lamina. Maturing sperm contain the histone deacetylase enzymes HDAC1 and HDAC3, suggesting the NuRD complex may drive some of these changes. Finally, using Western blotting, we detected changes in chromatin modifications between caput and caudal sperm isolated from rams (Ovis aries), inferring changes in histone modifications are a shared feature of mammalian epididymal maturation. Conclusions These data extend our understanding of germline programming and reveal that, in addition to trafficking noncoding RNAs, changes in histone posttranslational modifications are a core feature of epididymal maturation.

Patients with polyclonal hepatocellular carcinoma are at a high risk of early recurrence and have a poor recurrence-free survival period

Background/purpose of the study Tumor heterogeneity based on copy number variations is associated with the evolution of cancer and its clinical grade. Clonal composition (CC) represents the number of clones based on the distribution of B-allele frequency (BAF) obtained from a genome-wide single nucleotide polymorphism (SNP) array. A higher CC number represents a high degree of heterogeneity. We hypothesized and evaluated that the CC number in hepatocellular carcinoma (HCC) tissues might be associated with the clinical outcomes of patients. Methods Somatic mutation, whole transcriptome, and CC number based on copy number variations of 36 frozen tissue samples of operably resected HCC tissues were analyzed by targeted deep sequencing, transcriptome analysis, and SNP array. Results The samples were classified into the heterogeneous tumors as poly-CC (n = 26) and the homogeneous tumors as mono-CC (n = 8). The patients with poly-CC had a higher rate of early recurrence and a significantly shorter recurrence-free survival period than the mono-CC patients (7.0 months vs. not reached, p = 0.0084). No differences in pathogenic non-synonymous mutations, such as TP53, were observed between the two groups when targeted deep sequencing was applied. A transcriptome analysis showed that cell cycle-related pathways were enriched in the poly-CC tumors, compared to the mono-CC tumors. Poly-CC HCC is highly proliferative and has a high risk of early recurrence. Conclusion CC is a possible candidate biomarker for predicting the risk of early postoperative recurrence and warrants further investigation.

A Review on the Role of miR-149-5p in the Carcinogenesis

miR-149 is an miRNA with essential roles in carcinogenesis. This miRNA is encoded by the MIR149 gene on 2q37.3. The miR-149 hairpin produces miR-149-5p and miR-149-3p, which are the “guide” and the sister “passenger” strands, respectively. Deep sequencing experiments have shown higher prevalence of miR-149-5p compared with miR-149-3p. Notably, both oncogenic and tumor suppressive roles have been reported for miR-149-5p. In this review, we summarize the impact of miR-149-5p in the tumorigenesis and elaborate mechanisms of its involvement in this process in a variety of neoplastic conditions based on three lines of evidence, i.e., in vitro, in vivo and clinical settings.

Serology and Amplicon Deep Sequencing of MSP1-Block 2 Haplotypes as New Tools for Plasmodium Vivax Infections

Background: Relapses of Plasmodium vivax (P. vivax) infections are major causes of malaria morbidity, and tools for distinguishing relapses from reinfections are needed in malaria endemic areas. Methods: Herein, a panel of plasmas of 72 P. vivax-infected pregnant women, of whom 31 had had at least a recurrence of P. vivax infection, was used in a serology for IgM and IgG against 6 P. vivax-merozoite surface protein-1 (P. vivax-MSP1-Block 2) haplotype-specific peptides, in order to identify re-expositions to same haplotypes in the recurrences during the pregnancy. In parallel, we used the amplicon deep sequencing (ADS) with P. vivax-MSP1-Block 2 amplicons of the in eight blood samples of non-pregnant P. vivax-infected patients to identify multi or monoclonal infections based on MSP1-Block-2 haplotypes, and to quantify the reads of different haplotypes between those with multiclonal infections. We synthetized a new panel of overlapping peptides mapping each one of the six P. vivax-MSP1-Block 2 haplotypes and we validated with new IgM and IgG serology. Results: Most pregnant women presented IgM that recognized more than one peptide, thus indicating multiple infections by P. vivax-MSP1-Block 2 haplotypes. The same IgM anti-peptides remained in several women in the recurrent episodes most likely indicates re-exposure to the same haplotype of MSP1 Block 2. The IgG reactivity the IgM to IgG switch were low. The ADS using next-generation sequencing (NGS) identified multi- and monoinfection by P. vivax-MSP1-Block 2 haplotypes. Of eight patients, two of them had had the first P. vivax-infection. Monoinfections with P. vivax-MSP1-Block 2 haplotypes were observed in two prime-infected patients and three of patients with previous malaria. In all P. vivax-MSP1-Block 2 haplotype-monoinfected patients, the reactivity of IgM was observed only against overlapped peptides of the same haplotype detected in ADS, while for IgG, no reactivity was observed for any of the peptides of the same haplotype or the others.We were able to identify multiclonal infections through three haplotypes of P. vivax MSP1 Block 2 in three remaining patients, among which, there was always one majority haplotype that predominated with more of 95% of high-quality reads. The levels of haplotype-specific IgM in the serology correlated with the read ratios of each haplotype, but IgG levels not, including in one of the multiclonal infections, a minority haplotype was recognized with higher levels of IgG than that of the majority one. Conclusion: Our findings suggest that the combination of ADS and serology for P. vivax-MSP1-Block 2 haplotypes may be used as a new tool for distinguishing reinfections from relapses in malaria.

Early detection and improved genomic surveillance of SARS-CoV-2 variants from deep sequencing data

In the definition of fruitful strategies to contrast the worldwide diffusion of SARS-CoV-2, maximum efforts must be devoted to the early detection of dangerous variants. An effective help to this end is granted by the analysis of deep sequencing data of viral samples, which are typically discarded after the creation of consensus sequences. Indeed, only with deep sequencing data it is possible to identify intra-host low-frequency mutations, which are a direct footprint of mutational processes that may eventually lead to the origination of functionally advantageous variants. Accordingly, a timely and statistically robust identification of such mutations might inform political decision-making with significant anticipation with respect to standard analyses based on consensus sequences. To support our claim, we here present the largest study to date of SARS-CoV-2 deep sequencing data, which involves 220,788 high quality samples, collected over 20 months from 137 distinct studies. Importantly, we show that a relevant number of spike and nucleocapsid mutations of interest associated to the most circulating variants, including Beta, Delta and Omicron, might have been intercepted several months in advance, possibly leading to different public-health decisions. In addition, we show that a refined genomic surveillance system involving high- and low-frequency mutations might allow one to pinpoint possibly dangerous emerging mutation patterns, providing a data-driven automated support to epidemiologists and virologists.