Mutational profile of previously treated chronic lymphocytic leukemia patients progressing on acalabrutinib or ibrutinib.

Chronic lymphocytic leukemia (CLL) progression during Bruton tyrosine kinase (BTK) inhibitor treatment is typically characterized by emergent B-cell receptor pathway mutations. Using peripheral blood samples from relapsed/refractory CLL patients in ELEVATE-RR (NCT02477696) (median 2 prior therapies), we report clonal evolution data for patients progressing on acalabrutinib or ibrutinib (median follow-up 41 months). Paired (baseline and progression) samples were available for 47 (excluding 1 Richter) acalabrutinib-treated and 30 (excluding 6 Richter) ibrutinib-treated patients. At progression, emergent BTK mutations were observed in 31 (66%) acalabrutinib-treated and 11 (37%) ibrutinib-treated patients (median variant allele fraction [VAF]: 16.1% vs 15.6%). BTK C481S mutations were most common in both groups; T474I (n = 9; 8 co-occurring with C481) and the novel E41V mutation within the pleckstrin homology domain of BTK (n = 1) occurred with acalabrutinib, while neither mutation occurred with ibrutinib. L528W and A428D co-mutations presented in one ibrutinib-treated patient. Pre-existing TP53 mutations were present in 25 (53.2%) acalabrutinib-treated and 16 (53.3%) ibrutinib-treated patients at screening. Emergent TP53 mutations occurred with acalabrutinib and ibrutinib (13% vs 7%; median VAF: 6.0% vs 37.3%, respectively). Six acalabrutinib-treated patients and one ibrutinib-treated patient had emergent TP53/BTK co-mutations. Emergent PLCG2 mutations occurred in 3 (6%) acalabrutinib-treated and 6 (20%) ibrutinib-treated patients. One acalabrutinib-treated patient and 4 ibrutinib-treated patients had emergent BTK/PLCG2 co-mutations. While common BTK C481 mutations were observed with both treatments, patterns of mutation and co-mutation frequency, mutation VAF, and uncommon BTK variants varied with acalabrutinib (T474I and E41V) and ibrutinib (L528W, A428D) in this patient population.

Composting post-anaerobic digestion for emerging contaminant biodegradation: Impacts of operating conditions.

Sustainable manure management technologies are needed, and combining anaerobic digestion (AD) for energy generation and aerobic composting (AC) to stabilize digestate and remove emerging contaminants (ECs), including veterinary pharmaceuticals and steroid hormones, is promising. This study identified post-AD, AC operating conditions that maximized degradation of study ECs, expected to be present in cattle manure digested using treated municipal wastewater as the water source. Study ECs included sulfamethoxazole (SMX), chlortetracycline (CTC), oxytetracycline (OTC), estrone (E1), and naproxen (NPX). Composting conditions were simulated in bench-scale reactors, with microorganisms from digestate produced in an AD system (25L scale), by varying temperatures, pH, and carbon source compositions (representing food waste/manure co-digestion with different residence times). Results indicate maximum SMX biodegradation occurred at 35°C, pH 7, and with high levels of easily degradable carbon (≥99%, 99%, and 98%), and maximum E1 biodegradation occurred at 35°C, and with low levels of easily degradable carbon (≥97% and 99%). Abiotic degradation was responsible for the nearly complete removal of tetracyclines under all conditions and for partial degradation of NPX (between 20% and 48%). Microorganisms originating from the AD system putatively capable of SMX and E1 biodegradation, or of contributing to biodegradation during the AC phase, were identified, including phylotypes previously shown to biodegrade SMX (Brevundimonas and Alcaligenes).

Pulling needles out of a haystack: Subtractive Community Metatranscriptomics retrieves anaerobic o-xylene degradation pathway genes out of a mixed microbial culture.

For many contaminants, biomarker genes are unknown or assays are unavailable, and most biomarker assays target the first pathway step. Herein, we obtained sequences for all of the genes in a previously hypothesized o-xylene degradation pathway based on similarities to analogous genes in a known toluene degradation pathway. Comparative metatranscriptomics resulted in sequences for genes annotated as bssA, bbsEF, bbsCD, and bbsB, while genes for bbsG and bbsH were notably missing. Prokaryotic Suppressive Subtractive Hybridization PCR cDNA Subtraction (Prokaryotic SSH-PCR cDNA Subtraction) was applied for the first time to a mixed-species microbiome to enrich abundances of genes up-regulated during o-xylene degradation prior to metatranscriptomic sequencing. The subtracted metatranscriptome was sequenced using the MinION; this approach was highly effective at retrieving sequences for biodegradation genes including the previously missing bbsG and bbsH. Reverse transcription quantitative PCR (RT-qPCR) analysis confirmed up-regulation. Thus, data reported herein lend credence to the previously hypothesized anaerobic o-xylene degradation pathway, and new biomarker assays are presented. A novel biomarker development tool for mixed species systems, Subtractive Community Metatranscriptomics (SCM), is demonstrated.

Busulfan dose Recommendation in Inherited Metabolic Disorders: Population Pharmacokinetic Analysis.

Busulfan is a commonly used alkylating agent in the conditioning regimens of hematopoietic cell transplantation (HCT). Population pharmacokinetic (popPK) models enable description of busulfan PK and optimization of exposure, which leads to improvement of event-free survival after HCT. Prior busulfan popPK analysis has been limited by small numbers in patients with inherited metabolic disorders (IMD). The primary objective was to characterize population PK of busulfan in a large cohort of children and young adults undergoing HCT for IMD. PopPK analysis of busulfan drug concentrations was performed using data from 78 patients with IMD who received intravenous busulfan (every 24 hours, 4 doses) as part of pretransplantation combination chemotherapy. The final model for busulfan drug clearance was used to estimate individual doses aimed to achieve a target cumulative area under the curve (cAUC) of 80 to 100 mg · h/L. We then compared the probability of cAUC within the range of 80 to 100 mg · h/L by the developed dosing regimen versus conventional regimen. A 1-compartment, linear elimination model best described the PK of busulfan. Significant covariates demonstrated to affect busulfan clearance included total body weight and the time (in days) from busulfan infusion start. The probability of target cAUC attainment by the developed dosing versus the conventional dosing were 47% versus 43% for body weight <12 kg, and 48% versus 36% for body weight ≥12 kg. We described population PK of intravenous busulfan in a large IMD cohort. The proposed dosing regimen based on the developed model can improve the target cAUC attainment of busulfan for IMD.

Advanced methods for RNA recovery from petroleum impacted soils.

Microbially-mediated hydrocarbon degradation is well documented. However, how these microbial processes occur in complex subsurface petroleum impacted systems remains unclear, and this knowledge is needed to guide technologies to enhance microbial degradation effectively. Analysis of RNA derived from soils impacted by petroleum liquids would allow for analysis of active microbial communities, and a deeper understanding of the dynamic biochemistry occurring during site remediation. However, RNA analysis in soils impacted with petroleum liquids is challenging due to: (A) RNA being inherently unstable, and (B) petroleum impacted soils containing problematic levels of polymerase chain reaction (PCR) inhibitors that must be removed to yield high-purity RNA for downstream analysis. A previously published soil wash pretreatment step and a commercially available DNA extraction kit protocol were combined and modified to be able to purify RNA from soils containing petroleum liquids.•A key modification involved reformulation of the pretreatment solution via replacing water as the diluent with a commercially-available RNA preservation solution.•Methods were developed and demonstrated using cryogenically preserved soils from three former petroleum refineries. Results showed the new soil washing approach had no adverse effects on RNA recovery but did improve RNA quality, by PCR inhibitor removal, which in turn allows for characterization of active microbial communities present in petroleum impacted soils.•In summary, our method for extracting RNA from petroleum-impacted soils provides a promising new tool for resolving metabolic processes at sites as they progress toward restoration via natural and/or engineered remediation.

Wastewater Surveillance for SARS-CoV-2 on College Campuses: Initial Efforts, Lessons Learned, and Research Needs.

Wastewater surveillance for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging approach to help identify the risk of a coronavirus disease (COVID-19) outbreak. This tool can contribute to public health surveillance at both community (wastewater treatment system) and institutional (e.g., colleges, prisons, and nursing homes) scales. This paper explores the successes, challenges, and lessons learned from initial wastewater surveillance efforts at colleges and university systems to inform future research, development and implementation. We present the experiences of 25 college and university systems in the United States that monitored campus wastewater for SARS-CoV-2 during the fall 2020 academic period. We describe the broad range of approaches, findings, resources, and impacts from these initial efforts. These institutions range in size, social and political geographies, and include both public and private institutions. Our analysis suggests that wastewater monitoring at colleges requires consideration of local information needs, sewage infrastructure, resources for sampling and analysis, college and community dynamics, approaches to interpretation and communication of results, and follow-up actions. Most colleges reported that a learning process of experimentation, evaluation, and adaptation was key to progress. This process requires ongoing collaboration among diverse stakeholders including decision-makers, researchers, faculty, facilities staff, students, and community members.

Wastewater surveillance for SARS-CoV-2 on college campuses: Initial efforts, lessons learned and research needs.

Background: Wastewater surveillance for SARS-CoV-2 is an emerging approach to help identify the risk of a COVID-19 outbreak. This tool can contribute to public health surveillance at both community (wastewater treatment system) and institutional (e.g., colleges, prisons, nursing homes) scales. Objectives: This research aims to understand the successes, challenges, and lessons learned from initial wastewater surveillance efforts at colleges and university systems to inform future research, development and implementation. Methods: This paper presents the experiences of 25 college and university systems in the United States that monitored campus wastewater for SARS-CoV-2 during the fall 2020 academic period. We describe the broad range of approaches, findings, resource needs, and lessons learned from these initial efforts. These institutions range in size, social and political geographies, and include both public and private institutions. Discussion: Our analysis suggests that wastewater monitoring at colleges requires consideration of information needs, local sewage infrastructure, resources for sampling and analysis, college and community dynamics, approaches to interpretation and communication of results, and follow-up actions. Most colleges reported that a learning process of experimentation, evaluation, and adaptation was key to progress. This process requires ongoing collaboration among diverse stakeholders including decision-makers, researchers, faculty, facilities staff, students, and community members.

Inoculum microbiome composition impacts fatty acid product profile from cellulosic feedstock.

Conversion of organic wastes to fatty acids rather than methane through anaerobic digestion-based technologies has considerable promise. However, the relationships between microbiome structure and fatty acids produced from cellulosic feedstocks are not well understood. This study investigated the nature of those relationships for anaerobic digester sludge, bison rumen, and cattle rumen inocula grown on cellulose. Acetic acid production was highest in anaerobic sludge reactors, while propionic acid production was highest in cattle rumen reactors. Butyric and pentanoic acid were produced at the highest rates in bison rumen before Day 5. Reactor microbiomes remained distinct, despite identical operating conditions. Novel associations linked Alistipes with butyric acid production and Eubacterium nodatum and Clostridiales bacterium with pentanoic acid production. This study provides new insights into the ability of microbiomes to convert cellulose to different fatty acid mixtures and adds impetus for the rewiring of anaerobic digestion to generate high-value products.

Advancing biomarkers for anaerobic o-xylene biodegradation via metagenomic analysis of a methanogenic consortium.

Quantifying functional biomarker genes and their transcripts provides critical lines of evidence for contaminant biodegradation; however, accurate quantification depends on qPCR primers that contain no, or minimal, mismatches with the target gene. Developing accurate assays has been particularly challenging for genes encoding fumarate-adding enzymes (FAE) due to the high level of genetic diversity in this gene family. In this study, metagenomics applied to a field-derived, o-xylene-degrading methanogenic consortium revealed genes encoding FAE that would not be accurately quantifiable by any previously available PCR assays. Sequencing indicated that a gene similar to the napthylmethylsuccinate synthase gene (nmsA) was most abundant, although benzylsuccinate synthase genes (bssA) also were present along with genes encoding alkylsuccinate synthase (assA). Upregulation of the nmsA-like gene was observed during o-xylene degradation. Protein homology modeling indicated that mutations in the active site, relative to a BssA that acts on toluene, increase binding site volume and accessibility, potentially to accommodate the relatively larger o-xylene. The new nmsA-like gene was also detected at substantial concentrations at field sites with a history of xylene contamination.

Impact of primary carbon sources on microbiome shaping and biotransformation of pharmaceuticals and personal care products.

Knowledge of the conditions that promote the growth and activity of pharmaceutical and personal care product (PPCP)-degrading microorganisms within mixed microbial systems are needed to shape microbiomes in biotreatment reactors and manage process performance. Available carbon sources influence microbial community structure, and specific carbon sources could potentially be added to end-of-treatment train biotreatment systems (e.g., soil aquifer treatment [SAT]) to select for the growth and activity of a range of microbial phylotypes that collectively degrade target PPCPs. Herein, the impacts of primary carbon sources on PPCP biodegradation and microbial community structure were explored to identify promising carbon sources for PPCP biotreatment application. Six types of primary carbon sources were investigated: casamino acids, two humic acid and peptone mixtures (high and low amounts of humic acid), molasses, an organic acids mixture, and phenol. Biodegradation was tracked for five PPCPs (diclofenac, 5-fluorouracil, gemfibrozil, ibuprofen, and triclosan). Primary carbon sources were found to differentially impact microbial community structures and rates and efficiencies of PPCP biotransformation. Of the primary carbon sources tested, casamino acids, organic acids, and phenol showed the fastest biotransformation; however, on a biomass-normalized basis, both humic acid-peptone mixtures showed comparable or superior biotransformation. By comparing microbial communities for the different primary carbon sources, abundances of unclassified Beijerinckiaceae, Beijerinckia, Sphingomonas, unclassified Sphingomonadaceae, Flavobacterium, unclassified Rhizobiales, and Nevskia were statistically linked with biotransformation of specific PPCPs.

Child's play: Harnessing play and curiosity motives to improve child handwashing in a humanitarian setting.

In humanitarian emergency settings there is need for low cost and rapidly deployable interventions to protect vulnerable children, in- and out-of-school, from diarrhoeal diseases. Handwashing with soap can greatly reduce diarrhoea but interventions specifically targeting children's handwashing behaviour in humanitarian settings have not been tested. Traditional children's handwashing promotion interventions have been school-focused, resource-intensive and reliant on health-based messaging. However, recent research from non-humanitarian settings and targeting adults suggests that theory-based behaviour change interventions targeting specific motives may be more effective than traditional handwashing interventions. In this proof-of-concept study we test, for the first time, the distribution of a modified soap bar, designed to appeal to the motives of play and curiosity, in a household-level, rapidly deployable, handwashing promotion intervention for older children in a humanitarian setting - an internally displaced persons camp in Iraqi Kurdistan. Out of five total blocks within the camp, one was assigned to intervention and one to control. 40 households from each assigned block were then randomly chosen for inclusion in the study and the practice of handwashing with soap at key times was measured at baseline and four weeks after intervention delivery. Children in intervention households received transparent soaps with embedded toys, delivered within a short, fun, and interactive household session with minimal, non-health-based, messaging. The control group received plain soap delivered in a short standard, health-based, hygiene promotion session. At the 4-week follow-up, children in the intervention group were 4 times more likely to wash their hands with soap after key handwashing occasions than expected in the counterfactual (if there had been no intervention) based on the comparison to children in the control group (adjusted RR = 3.94, 95% CI 1.59-9.79). We show that distributing soaps with toys embedded inside, in a rapidly deployable intervention, can improve child handwashing behaviour in a humanitarian emergency context. Further studies are needed to determine the longer-term behavioural and health impact of such an intervention when delivered at a greater scale in a humanitarian context.

Meta-proteomic analysis of protein expression distinctive to electricity-generating biofilm communities in air-cathode microbial fuel cells.

BACKGROUND: Bioelectrochemical systems (BESs) harness electrons from microbial respiration to generate power or chemical products from a variety of organic feedstocks, including lignocellulosic biomass, fermentation byproducts, and wastewater sludge. In some BESs, such as microbial fuel cells (MFCs), bacteria living in a biofilm use the anode as an electron acceptor for electrons harvested from organic materials such as lignocellulosic biomass or waste byproducts, generating energy that may be used by humans. Many BES applications use bacterial biofilm communities, but no studies have investigated protein expression by the anode biofilm community as a whole. RESULTS: To discover functional protein expression during current generation that may be useful for MFC optimization, a label-free meta-proteomics approach was used to compare protein expression in acetate-fed anode biofilms before and after the onset of robust electricity generation. Meta-proteomic comparisons were integrated with 16S rRNA gene-based community analysis at four developmental stages. The community composition shifted from dominance by aerobic Gammaproteobacteria (90.9 ± 3.3%) during initial biofilm formation to dominance by Deltaproteobacteria, particularly Geobacter (68.7 ± 3.6%) in mature, electricity-generating anodes. Community diversity in the intermediate stage, just after robust current generation began, was double that at the early stage and nearly double that of mature anode communities. Maximum current densities at the intermediate stage, however, were relatively similar (~ 83%) to those achieved by mature-stage biofilms. Meta-proteomic analysis, correlated with population changes, revealed significant enrichment of categories specific to membrane and transport functions among proteins from electricity-producing biofilms. Proteins detected only in electricity-producing biofilms were associated with gluconeogenesis, the glyoxylate cycle, and fatty acid ß-oxidation, as well as with denitrification and competitive inhibition. CONCLUSIONS: The results demonstrate that it is possible for an MFC microbial community to generate robust current densities while exhibiting high taxonomic diversity. Moreover, these data provide evidence to suggest that startup growth of air-cathode MFCs under conditions that promote the establishment of aerobic-anaerobic syntrophy may decrease startup times. This study represents the first investigation into protein expression of a complex BES anode biofilm community as a whole. The findings contribute to understanding of the molecular mechanisms at work during BES startup and suggest options for improvement of BES generation of bioelectricity from renewable biomass.

Ambient-temperature co-digestion of low-solids municipal and industrial waste mixtures: Insights from molecular analyses.

The performance of ambient temperature anaerobic co-digestion was investigated for mixtures of six substrates: canned tomato and salsa waste, portable toilet waste, septic tank waste, winery waste, beer and cider waste, and fats, oils, and grease (FOG). Laboratory semi-continuous reactor studies and molecular biological analyses revealed that beer/winery, and tomato/FOG/winery/beer mixtures resulted in the best performance in terms of biogas production (515 and 371 mL CH4/g VS, respectively) and methanogenic populations. A portable toilet/septage mixture resulted in the overall poorest performance and inhibition of microbial activity was evident. Average methane content was ~70% for all mixtures tested. The findings of this study reveal that healthy methanogen populations were present, further supporting the feasibility of biogas production via the novel feedstock mixtures in ambient temperature lagoons. Implications: Disposal of septic tank waste and other high chemical oxygen demand (COD) 10 industrial food processing waste at a small wastewater treatment plant is uncommon, because it can upset the treatment process and requires additional power for treatment. Ambient-temperature covered lagoon digesters can be an alternative low-cost technology for co-digestion of these recalcitrant waste streams while generating bioenergy. The results of this study demonstrated that there is potential for implementation of unheated covered lagoon digester systems 15 for conversion of liquid wastes for production of renewable biomethane while eliminating the need to treat these wastes at a wastewater treatment plant.

Differential Sensitivity of Wetland-Derived Nitrogen Cycling Microorganisms to Copper Nanoparticles.

Metallic nanoparticles (NPs), the most abundant nanomaterials in consumer and industrial products, are the most probable class to enter the environment. In this study, wetland-derived microcosms were incubated with copper nanoparticles (Cu-NP) and ionic CuCl2 to investigate acute (10 days) and chronic (100 days) exposure towards nitrogen cycling microorganisms. The microbial ecology of wetlands play a crucial role in balancing nitrogen in pristine environments as well as in areas impacted by high nutrient loads (e.g., at wastewater effluent discharges). Gene abundance and expression changes were monitored using the GeoChip 5.0 high throughput functional gene microarray and metatranscriptomic shotgun sequencing (RNA-seq), respectively. After 10 days, the Cu-NP impacted microbial communities experienced structural shifts within microorganisms associated with dissimilatory nitrogen reduction accompanied by lower nitrate removal as compared to the unexposed controls. By day 100, these differences were largely resolved and nitrate removal was similar to the unexposed control. Furthermore, the Cu-NP exposed microcosms tolerated copper and were more resilient and adaptive than the unexposed controls based on the abundance and expression of other functions, including electron transfer, metal homeostasis, and stress response. These findings suggest sudden influxes of Cu-NPs into wetland systems may impair nitrogen removal initially, but long-term microbial shifts and functional redundancy would promote the net flux of total nitrogen out of the wetlands.

Impact of inoculum sources on biotransformation of pharmaceuticals and personal care products.

Limited knowledge of optimal microbial community composition for PPCP biotreatment, and of the microbial phylotypes that drive biotransformation within mixed microbial communities, has hindered the rational design and operation of effective and reliable biological PPCP treatment technologies. Herein, bacterial community composition was investigated as an isolated variable within batch biofilm reactors via comparison of PPCP removals for three distinct inocula. Inocula pre-acclimated to model PPCPs were derived from activated sludge (AS), ditch sediment historically-impacted by wastewater treatment plant effluent (Sd), and material from laboratory-scale soil aquifer treatment (SAT) columns. PPCP removals were found to be substantially higher for AS- and Sd-derived inocula compared to the SAT-derived inocula despite comparable biomass. Removal patterns differed among the 6 model compounds examined (diclofenac, 5-fluorouracil, gabapentin, gemfibrozil, ibuprofen, and triclosan) indicating differences in biotransformation mechanisms. Sphingomonas, Beijerinckia, Methylophilus, and unknown Cytophagaceae were linked with successful PPCP biodegradation via next-generation sequencing of 16S rRNA genes over time. Results indicate the criticality of applying engineering approaches to control bacterial community compositions in biotreatment systems.

Enhanced anaerobic digestion performance via combined solids- and leachate-based hydrolysis reactor inoculation.

Suboptimal conditions in anaerobic digesters (e.g., presence of common inhibitors ammonia and salinity) limit waste hydrolysis and lead to unstable performance and process failures. Application of inhibitor-tolerant inocula improves hydrolysis, but approaches are needed to establish and maintain these desired waste-hydrolyzing bacteria in high-solids reactors. Herein, performance was compared for leach bed reactors (LBRs) seeded with unacclimated or acclimated inoculum (0-60% by mass) at start-up and over long-term operation. High quantities of inoculum (∼60%) increase waste hydrolysis and are beneficial at start-up or when inhibitors are increasing. After start-up (∼112days) with high inoculum quantities, leachate recirculation leads to accumulation of inhibitor-tolerant hydrolyzing bacteria in leachate. During long-term operation, low inoculum quantities (∼10%) effectively increase waste hydrolysis relative to without solids-derived inoculum. Molecular analyses indicated that combining digested solids with leachate-based inoculum doubles quantities of Bacteria contacting waste over a batch and supplies additional desirable phylotypes Bacteriodes and Clostridia.

Comparison of bacterial and archaeal communities in depth-resolved zones in an LNAPL body.

Advances in our understanding of the microbial ecology at sites impacted by light non-aqueous phase liquids (LNAPLs) are needed to drive development of optimized bioremediation technologies, support longevity models, and develop culture-independent molecular tools. In this study, depth-resolved characterization of geochemical parameters and microbial communities was conducted for a shallow hydrocarbon-impacted aquifer. Four distinct zones were identified based on microbial community structure and geochemical data: (i) an aerobic, low-contaminant mass zone at the top of the vadose zone; (ii) a moderate to high-contaminant mass, low-oxygen to anaerobic transition zone in the middle of the vadose zone; (iii) an anaerobic, high-contaminant mass zone spanning the bottom of the vadose zone and saturated zone; and (iv) an anaerobic, low-contaminant mass zone below the LNAPL body. Evidence suggested that hydrocarbon degradation is mediated by syntrophic fermenters and methanogens in zone III. Upward flux of methane likely contributes to promoting anaerobic conditions in zone II by limiting downward flux of oxygen as methane and oxygen fronts converge at the top of this zone. Observed sulfate gradients and microbial communities suggested that sulfate reduction and methanogenesis both contribute to hydrocarbon degradation in zone IV. Pyrosequencing revealed that Syntrophus- and Methanosaeta-related species dominate bacterial and archaeal communities, respectively, in the LNAPL body below the water table. Observed phylotypes were linked with in situ anaerobic hydrocarbon degradation in LNAPL-impacted soils.

Immunoglobulin transcript sequence and somatic hypermutation computation from unselected RNA-seq reads in chronic lymphocytic leukemia.

Immunoglobulins (Ig) are produced by B lymphocytes as secreted antibodies or as part of the B-cell receptor. There is tremendous diversity of potential Ig transcripts (>1 × 10(12)) as a result of hundreds of germ-line gene segments, random nucleotide incorporation during joining of gene segments into a complete transcript, and the process of somatic hypermutation at individual nucleotides. This recombination and mutation process takes place in the maturing B cell and is responsible for the diversity of potential epitope recognition. Cancers arising from mature B cells are characterized by clonal production of Ig heavy (IGH@) and light chain transcripts, although whether the sequence has undergone somatic hypermutation is dependent on the maturation stage at which the neoplastic clone arose. Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults and arises from a mature B cell with either mutated or unmutated IGH@ transcripts, the latter having worse prognosis and the assessment of which is routinely performed in the clinic. Currently, IGHV mutation status is assessed by Sanger sequencing and comparing the transcript to known germ-line genes. In this paper, we demonstrate that complete IGH@ V-D-J sequences can be computed from unselected RNA-seq reads with results equal or superior to the clinical procedure: in the only discordant case, the clinical transcript was out-of-frame. Therefore, a single RNA-seq assay can simultaneously yield gene expression profile, SNP and mutation information, as well as IGHV mutation status, and may one day be performed as a general test to capture multidimensional clinically relevant data in CLL.