The evolution of synaptic and cognitive capacity: Insights from the nervous system transcriptome of Aplysia.

The gastropod mollusk Aplysia is an important model for cellular and molecular neurobiological studies, particularly for investigations of molecular mechanisms of learning and memory. We developed an optimized assembly pipeline to generate an improved Aplysia nervous system transcriptome. This improved transcriptome enabled us to explore the evolution of cognitive capacity at the molecular level. Were there evolutionary expansions of neuronal genes between this relatively simple gastropod Aplysia (20,000 neurons) and Octopus (500 million neurons), the invertebrate with the most elaborate neuronal circuitry and greatest behavioral complexity? Are the tremendous advances in cognitive power in vertebrates explained by expansion of the synaptic proteome that resulted from multiple rounds of whole genome duplication in this clade? Overall, the complement of genes linked to neuronal function is similar between Octopus and Aplysia. As expected, a number of synaptic scaffold proteins have more isoforms in humans than in Aplysia or Octopus. However, several scaffold families present in mollusks and other protostomes are absent in vertebrates, including the Fifes, Lev10s, SOLs, and a NETO family. Thus, whereas vertebrates have more scaffold isoforms from select families, invertebrates have additional scaffold protein families not found in vertebrates. This analysis provides insights into the evolution of the synaptic proteome. Both synaptic proteins and synaptic plasticity evolved gradually, yet the last deuterostome-protostome common ancestor already possessed an elaborate suite of genes associated with synaptic function, and critical for synaptic plasticity.

Accumulation of endosymbiont genomes in an insect autosome followed by endosymbiont replacement.

Eukaryotic genomes can acquire bacterial DNA via lateral gene transfer (LGT).1 A prominent source of LGT is Wolbachia,2 a widespread endosymbiont of arthropods and nematodes that is transmitted maternally through female germline cells.3,4 The DNA transfer from the Wolbachia endosymbiont wAna to Drosophila ananassae is extensive5-7 and has been localized to chromosome 4, contributing to chromosome expansion in this lineage.6 As has happened frequently with claims of bacteria-to-eukaryote LGT, the contribution of wAna transfers to the expanded size of D. ananassae chromosome 4 has been specifically contested8 owing to an assembly where Wolbachia sequences were classified as contaminants and removed.9 Here, long-read sequencing with DNA from a Wolbachia-cured line enabled assembly of 4.9 Mbp of nuclear Wolbachia transfers (nuwts) in D. ananassae and a 24-kbp nuclear mitochondrial transfer. The nuwts are <8,000 years old in at least two locations in chromosome 4 with at least one whole-genome integration followed by rapid extensive duplication of most of the genome with regions that have up to 10 copies. The genes in nuwts are accumulating small indels and mobile element insertions. Among the highly duplicated genes are cifA and cifB, two genes associated with Wolbachia-mediated Drosophila cytoplasmic incompatibility. The wAna strain that was the source of nuwts was subsequently replaced by a different wAna endosymbiont. Direct RNA Nanopore sequencing of Wolbachia-cured lines identified nuwt transcripts, including spliced transcripts, but functionality, if any, remains elusive.

Highly Specialized Carbohydrate Metabolism Capability in Bifidobacterium Strains Associated with Intestinal Barrier Maturation in Early Preterm Infants.

"Leaky gut," or high intestinal barrier permeability, is common in preterm newborns. The role of the microbiota in this process remains largely uncharacterized. We employed both short- and long-read sequencing of the 16S rRNA gene and metagenomes to characterize the intestinal microbiome of a longitudinal cohort of 113 preterm infants born between 240/7 and 326/7 weeks of gestation. Enabled by enhanced taxonomic resolution, we found that a significantly increased abundance of Bifidobacterium breve and a diet rich in mother's breastmilk were associated with intestinal barrier maturation during the first week of life. We combined these factors using genome-resolved metagenomics and identified a highly specialized genetic capability of the Bifidobacterium strains to assimilate human milk oligosaccharides and host-derived glycoproteins. Our study proposes mechanistic roles of breastmilk feeding and intestinal microbial colonization in postnatal intestinal barrier maturation; these observations are critical toward advancing therapeutics to prevent and treat hyperpermeable gut-associated conditions, including necrotizing enterocolitis (NEC). IMPORTANCE Despite improvements in neonatal intensive care, necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality. "Leaky gut," or intestinal barrier immaturity with elevated intestinal permeability, is the proximate cause of susceptibility to NEC. Early detection and intervention to prevent leaky gut in "at-risk" preterm neonates are critical for decreasing the risk of potentially life-threatening complications like NEC. However, the complex interactions between the developing gut microbial community, nutrition, and intestinal barrier function remain largely uncharacterized. In this study, we reveal the critical role of a sufficient breastmilk feeding volume and the specialized carbohydrate metabolism capability of Bifidobacterium in the coordinated postnatal improvement of the intestinal barrier. Determining the clinical and microbial biomarkers that drive the intestinal developmental disparity will inform early detection and novel therapeutic strategies to promote appropriate intestinal barrier maturation and prevent NEC and other adverse health conditions in preterm infants.

Successful Profiling of Plasmodium falciparum var Gene Expression in Clinical Samples via a Custom Capture Array.

var genes encode Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche's SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional >4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var-like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis. IMPORTANCE Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. Identifying these expressed antigens is critical to understanding the development of severe malarial disease. However, clinical samples contain limited amounts of parasite genetic material, a challenge for sequencing efforts further compounded by the extreme diversity of the parasite surface antigens. We present a method that enriches for these antigen sequences in clinical samples using a custom capture array, requiring minimal processing in the field. While our results are focused on the malaria parasite Plasmodium falciparum, this approach has broad applicability to other highly diverse antigens from other parasites and pathogens such as those that cause giardiasis and leishmaniasis.

X-treme loss of sequence diversity linked to neo-X chromosomes in filarial nematodes.

The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected (πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication.

Evaluation of a high-throughput, cost-effective Illumina library preparation kit.

Library preparation for high-throughput sequencing applications is a critical step in producing representative, unbiased sequencing data. The iGenomX Riptide High Throughput Rapid Library Prep Kit purports to provide high-quality sequencing data with lower costs compared to other Illumina library kits. To test these claims, we compared sequence data quality of Riptide libraries to libraries constructed with KAPA Hyper and NEBNext Ultra. Across several single-source genome samples, mapping performance and de novo assembly of Riptide libraries were similar to conventional libraries prepared with the same DNA. Poor performance of some libraries resulted in low sequencing depth. In particular, degraded DNA samples may be challenging to sequence with Riptide. There was little cross-well plate contamination with the overwhelming majority of reads belong to the proper source genomes. The sequencing of metagenome samples using different Riptide primer sets resulted in variable taxonomic assignment of reads. Increased adoption of the Riptide kit will decrease library preparation costs. However, this method might not be suitable for degraded DNA.

Comparison of long-read sequencing technologies in interrogating bacteria and fly genomes.

The newest generation of DNA sequencing technology is highlighted by the ability to generate sequence reads hundreds of kilobases in length. Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) have pioneered competitive long read platforms, with more recent work focused on improving sequencing throughput and per-base accuracy. We used whole-genome sequencing data produced by three PacBio protocols (Sequel II CLR, Sequel II HiFi, RS II) and two ONT protocols (Rapid Sequencing and Ligation Sequencing) to compare assemblies of the bacteria Escherichia coli and the fruit fly Drosophila ananassae. In both organisms tested, Sequel II assemblies had the highest consensus accuracy, even after accounting for differences in sequencing throughput. ONT and PacBio CLR had the longest reads sequenced compared to PacBio RS II and HiFi, and genome contiguity was highest when assembling these datasets. ONT Rapid Sequencing libraries had the fewest chimeric reads in addition to superior quantification of E. coli plasmids versus ligation-based libraries. The quality of assemblies can be enhanced by adopting hybrid approaches using Illumina libraries for bacterial genome assembly or polishing eukaryotic genome assemblies, and an ONT-Illumina hybrid approach would be more cost-effective for many users. Genome-wide DNA methylation could be detected using both technologies, however ONT libraries enabled the identification of a broader range of known E. coli methyltransferase recognition motifs in addition to undocumented D. ananassae motifs. The ideal choice of long read technology may depend on several factors including the question or hypothesis under examination. No single technology outperformed others in all metrics examined.

Soluble Sema4D in Plasma of Head and Neck Squamous Cell Carcinoma Patients Is Associated With Underlying Non-Inflamed Tumor Profile.

Semaphorin 4D (Sema4D) is a glycoprotein that is expressed by several tumors and immune cells. It can function as a membrane bound protein or as a cleaved soluble protein (sSema4D). We sought to investigate the translational potential of plasma sSema4D as an immune marker in plasma of patients with head and neck squamous cell carcinoma (HNSCC). Paired peripheral blood and tumor tissue samples of 104 patients with HNSCC were collected at the same time point to allow for real time analysis. Scoring of the histological inflammatory subtype (HIS) was carried out using Sema4D immunohistochemistry on the tumor tissue. sSema4D was detected in plasma using direct ELISA assay. Defining elevated sSema4D as values above the 95th percentile in healthy controls, our data showed that sSema4D levels in plasma were elevated in 25.0% (95% CI, 16.7-34.9%) of the patients with HNSCC and showed significant association with HIS immune excluded (HIS-IE) (p = 0.007), Sema4D+ve tumor cells (TCs) (p = 0.018) and PD-L1+ve immune cells (ICs) (p = 0.038). A multi-variable logistic regression analysis showed that HIS was significantly (P = 0.004) associated with elevated sSema4D, an association not explained by available patient-level factors. Using the IO-360 nanoString platform, differential gene expression (DGE) analysis of 10 HNSCC tumor tissues showed that patients with high sSema4D in plasma (HsS4D) clustered as IFN-γ negative tumor immune signature and were mostly HIS-IE. The IC type in the HsS4D paired tumor tissue was predominantly myeloid, while the lymphoid compartment was higher in the low sSema4D (LsS4D). The Wnt signaling pathway was upregulated in the HsS4D group. Further analysis using the IO-360, 770 gene set, showed significant non-inflamed profile of the HsS4D tumors compared to the LsS4D. In conclusion, our data reveals an association between sSema4D and the histological inflammatory subtype.

Haplotype-resolved diverse human genomes and integrated analysis of structural variation.

Long-read and strand-specific sequencing technologies together facilitate the de novo assembly of high-quality haplotype-resolved human genomes without parent-child trio data. We present 64 assembled haplotypes from 32 diverse human genomes. These highly contiguous haplotype assemblies (average minimum contig length needed to cover 50% of the genome: 26 million base pairs) integrate all forms of genetic variation, even across complex loci. We identified 107,590 structural variants (SVs), of which 68% were not discovered with short-read sequencing, and 278 SV hotspots (spanning megabases of gene-rich sequence). We characterized 130 of the most active mobile element source elements and found that 63% of all SVs arise through homology-mediated mechanisms. This resource enables reliable graph-based genotyping from short reads of up to 50,340 SVs, resulting in the identification of 1526 expression quantitative trait loci as well as SV candidates for adaptive selection within the human population.

FADU: a Quantification Tool for Prokaryotic Transcriptomic Analyses.

Quantification tools for RNA sequencing (RNA-Seq) analyses are often designed and tested using human transcriptomics data sets, in which full-length transcript sequences are well annotated. For prokaryotic transcriptomics experiments, full-length transcript sequences are seldom known, and coding sequences must instead be used for quantification steps in RNA-Seq analyses. However, operons confound accurate quantification of coding sequences since a single transcript does not necessarily equate to a single gene. Here, we introduce FADU (Feature Aggregate Depth Utility), a quantification tool designed specifically for prokaryotic RNA-Seq analyses. FADU assigns partial count values proportional to the length of the fragment overlapping the target feature. To assess the ability of FADU to quantify genes in prokaryotic transcriptomics analyses, we compared its performance to those of eXpress, featureCounts, HTSeq, kallisto, and Salmon across three paired-end read data sets of (i) Ehrlichia chaffeensis, (ii) Escherichia coli, and (iii) the Wolbachia endosymbiont wBm. Across each of the three data sets, we find that FADU can more accurately quantify operonic genes by deriving proportional counts for multigene fragments within operons. FADU is available at https://github.com/IGS/FADUIMPORTANCE Most currently available quantification tools for transcriptomics analyses have been designed for human data sets, in which full-length transcript sequences, including the untranslated regions, are well annotated. In most prokaryotic systems, full-length transcript sequences have yet to be characterized, leading to prokaryotic transcriptomics analyses being performed based on only the coding sequences. In contrast to eukaryotes, prokaryotes contain polycistronic transcripts, and when genes are quantified based on coding sequences instead of transcript sequences, this leads to an increased abundance of improperly assigned ambiguous multigene fragments, specifically those mapping to multiple genes in operons. Here, we describe FADU, a quantification tool for prokaryotic RNA-Seq analyses designed to assign proportional counts with the purpose of better quantifying operonic genes while minimizing the pitfalls associated with improperly assigning fragment counts from ambiguous transcripts.

Comparative Analysis of Genome of Ehrlichia sp. HF, a Model Bacterium to Study Fatal Human Ehrlichiosis.

BACKGROUND: The genus Ehrlichia consists of tick-borne obligatory intracellular bacteria that can cause deadly diseases of medical and agricultural importance. Ehrlichia sp. HF, isolated from Ixodes ovatus ticks in Japan [also referred to as I. ovatus Ehrlichia (IOE) agent], causes acute fatal infection in laboratory mice that resembles acute fatal human monocytic ehrlichiosis caused by Ehrlichia chaffeensis. As there is no small laboratory animal model to study fatal human ehrlichiosis, Ehrlichia sp. HF provides a needed disease model. However, the inability to culture Ehrlichia sp. HF and the lack of genomic information have been a barrier to advance this animal model. In addition, Ehrlichia sp. HF has several designations in the literature as it lacks a taxonomically recognized name. RESULTS: We stably cultured Ehrlichia sp. HF in canine histiocytic leukemia DH82 cells from the HF strain-infected mice, and determined its complete genome sequence. Ehrlichia sp. HF has a single double-stranded circular chromosome of 1,148,904 bp, which encodes 866 proteins with a similar metabolic potential as E. chaffeensis. Ehrlichia sp. HF encodes homologs of all virulence factors identified in E. chaffeensis, including 23 paralogs of P28/OMP-1 family outer membrane proteins, type IV secretion system apparatus and effector proteins, two-component systems, ankyrin-repeat proteins, and tandem repeat proteins. Ehrlichia sp. HF is a novel species in the genus Ehrlichia, as demonstrated through whole genome comparisons with six representative Ehrlichia species, subspecies, and strains, using average nucleotide identity, digital DNA-DNA hybridization, and core genome alignment sequence identity. CONCLUSIONS: The genome of Ehrlichia sp. HF encodes all known virulence factors found in E. chaffeensis, substantiating it as a model Ehrlichia species to study fatal human ehrlichiosis. Comparisons between Ehrlichia sp. HF and E. chaffeensis will enable identification of in vivo virulence factors that are related to host specificity, disease severity, and host inflammatory responses. We propose to name Ehrlichia sp. HF as Ehrlichia japonica sp. nov. (type strain HF), to denote the geographic region where this bacterium was initially isolated.

Complete Mitochondrial Genome Sequence of Mansonella perstans.

The 13,647-bp complete mitochondrial genome of Mansonella perstans was sequenced and is syntenic to the mitochondrial genome of Mansonella ozzardi Phylogenetic analysis of the mitochondrial genome is consistent with the known phylogeny of ONC5 group filarial nematodes.

Nearly Complete Genome Sequence of Brugia pahangi FR3.

Brugia pahangi is a zoonotic parasite that is closely related to human-infecting filarial nematodes. Here, we report the nearly complete genome of Brugia pahangi, including assemblies of four autosomes and an X chromosome, with only seven gaps. The Y chromosome is still not completely assembled.

Complete Genome Sequence of wBp, the Wolbachia Endosymbiont of Brugia pahangi FR3.

Lymphatic filariasis is a devastating disease caused by filarial nematode roundworms, which contain obligate Wolbachia endosymbionts. Here, we assembled the genome of wBp, the Wolbachia endosymbiont of the filarial nematode Brugia pahangi, from Illumina, Pacific Biosciences, and Oxford Nanopore data. The complete, circular genome is 1,072,967 bp.

Comparative Metagenome-Assembled Genome Analysis of "Candidatus Lachnocurva vaginae", Formerly Known as Bacterial Vaginosis-Associated Bacterium-1 (BVAB1).

Bacterial vaginosis-associated bacterium 1 (BVAB1) is an as-yet uncultured bacterial species found in the human vagina that belongs to the family Lachnospiraceae within the order Clostridiales. As its name suggests, this bacterium is often associated with bacterial vaginosis (BV), a common vaginal disorder that has been shown to increase a woman's risk for HIV, Chlamydia trachomatis, and Neisseria gonorrhoeae infections as well as preterm birth. BVAB1 has been further associated with the persistence of BV following metronidazole treatment, increased vaginal inflammation, and adverse obstetrics outcomes. There is no available complete genome sequence of BVAB1, which has made it difficult to mechanistically understand its role in disease. We present here a circularized metagenome-assembled genome (cMAG) of BVAB1 as well as a comparative analysis including an additional six metagenome-assembled genomes (MAGs) of this species. These sequences were derived from cervicovaginal samples of seven separate women. The cMAG was obtained from a metagenome sequenced with long-read technology on a PacBio Sequel II instrument while the others were derived from metagenomes sequenced on the Illumina HiSeq platform. The cMAG is 1.649 Mb in size and encodes 1,578 genes. We propose to rename BVAB1 to "Candidatus Lachnocurva vaginae" based on phylogenetic analyses, and provide genomic and metabolomic evidence that this candidate species may metabolize D-lactate, produce trimethylamine (one of the chemicals responsible for BV-associated odor), and be motile. The cMAG and the six MAGs are valuable resources that will further contribute to our understanding of the heterogeneous etiology of bacterial vaginosis.

Strains used in whole organism Plasmodium falciparum vaccine trials differ in genome structure, sequence, and immunogenic potential.

BACKGROUND: Plasmodium falciparum (Pf) whole-organism sporozoite vaccines have been shown to provide significant protection against controlled human malaria infection (CHMI) in clinical trials. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions. METHODS: Whole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generate de novo genome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequences in each strain relative to the reference 3D7 (a clone of NF54) genome. Strains were compared to each other and to a collection of clinical isolates (sequenced as part of this study or from public repositories) from South America, sub-Saharan Africa, and Southeast Asia. RESULTS: While few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains. These variants include SNPs, indels, and small structural variants that fall in regulatory and immunologically important regions, including transcription factors (such as PfAP2-L and PfAP2-G) and pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contributed to diversity in immunologically important regions of the genomes as detected through in silico CD8+ T cell epitope predictions. Of all heterologous strains, NF135.C10 had the highest number of unique predicted epitope sequences when compared to NF54. Comparison to global clinical isolates revealed that these four strains are representative of their geographic origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently formed subpopulations resistant to artemisinin-based therapies present in the Greater Mekong Sub-region. CONCLUSIONS: These results will assist in the interpretation of vaccine efficacy of whole-organism vaccines against homologous and heterologous CHMI.

Drug Repurposing of Bromodomain Inhibitors as Potential Novel Therapeutic Leads for Lymphatic Filariasis Guided by Multispecies Transcriptomics.

To better understand the transcriptomic interplay of organisms associated with lymphatic filariasis, we conducted multispecies transcriptome sequencing (RNA-Seq) on the filarial nematode Brugia malayi, its Wolbachia endosymbiont wBm, and its laboratory vector Aedes aegypti across the entire B. malayi life cycle. In wBm, transcription of the noncoding 6S RNA suggests that it may be a regulator of bacterial cell growth, as its transcript levels correlate with bacterial replication rates. For A. aegypti, the transcriptional response reflects the stress that B. malayi infection exerts on the mosquito with indicators of increased energy demand. In B. malayi, expression modules associated with adult female samples consistently contained an overrepresentation of genes involved in chromatin remodeling, such as the bromodomain-containing proteins. All bromodomain-containing proteins encoded by B. malayi were observed to be upregulated in the adult female, embryo, and microfilaria life stages, including 2 members of the bromodomain and extraterminal (BET) protein family. The BET inhibitor JQ1(+), originally developed as a cancer therapeutic, caused lethality of adult worms in vitro, suggesting that it may be a potential therapeutic that can be repurposed for treating lymphatic filariasis.IMPORTANCE The current treatment regimen for lymphatic filariasis is mostly microfilaricidal. In an effort to identify new drug candidates for lymphatic filariasis, we conducted a three-way transcriptomics/systems biology study of one of the causative agents of lymphatic filariasis, Brugia malayi, its Wolbachia endosymbiont wBm, and its vector host Aedes aegypti at 16 distinct B. malayi life stages. B. malayi upregulates the expression of bromodomain-containing proteins in the adult female, embryo, and microfilaria stages. In vitro, we find that the existing cancer therapeutic JQ1(+), which is a bromodomain and extraterminal protein inhibitor, has adulticidal activity in B. malayi.

Intratumor genetic heterogeneity in squamous cell carcinoma of the oral cavity.

BACKGROUND: We sought to evaluate intratumor heterogeneity in squamous cell carcinoma of the oral cavity (OCC) and specifically determine the effect of physical separation and histologic differentiation within the same tumor. METHODS: We performed whole exome sequencing on five biopsy sites-two from well-differentiated, two from poorly differentiated regions, and one from normal parenchyma-from five primary OCC specimens. RESULTS: We found high levels of intratumor heterogeneity and, in four primary tumors, identified only 0 to 2 identical mutations in all subsites. We found that the heterogeneity inversely correlated with physical separation and that pairs of well-differentiated samples were more similar to each other than analogous poorly differentiated specimens. Only TP53 mutations, but not other purported "driver mutations" in head and neck squamous cell carcinoma, were found in multiple biopsy sites. CONCLUSION: These data highlight the challenges to characterization of the mutational landscape of OCC with single site biopsy and have implications for personalized medicine.

Multispecies Transcriptomics Data Set of Brugia malayi, Its Wolbachia Endosymbiont wBm, and Aedes aegypti across the B. malayi Life Cycle.

Here, we present a comprehensive transcriptomics data set of Brugia malayi, its Wolbachia endosymbiont wBm, and its vector host. This study samples from 16 stages across the entire B. malayi life cycle, including stage 1 through 4 larvae, adult males and females, embryos, immature microfilariae, and mature microfilariae.

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses.

Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r2 = 0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2 = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies.