Construction Of Gene Libraries Research Articles

Fast and precise multi-site mutagenesis on linear DNA fragments

The effectiveness and scalability of site-directed mutagenesis are constrained by the limited number of mutations and the intricate cloning process required for isolation of the target sequence. Here, we present a method for precise introduction of multiple non-contiguous mutations. The mutant strands are collected through one specially designed magnetic bead separation in alkaline conditions, efficiently removing their complementary partner strands with the original sequences. In a proof-of-concept test, a green fluorescent protein (GFP) was simultaneously mutated in 1–3 specific amino acids, successfully shifting its fluorescence spectrum. The precise mutation rates for single-, double- and triple-site mutations reached 100%, 76% and 70%, respectively. This multiple non-contiguous mutagenesis method may offer a fast and cost-effective approach for customizable construction of gene library.

Construction and Application of Cultural Gene Library of Ancestral Hall in Canton Region

Construction and Application of Cultural Gene Library of Ancestral Hall in Canton Region

Discovery and mechanism explanation of a novel green biocatalyst esterase Bur01 from Burkholderia ambifaria for ester synthesis under aqueous phase

Biocatalyst catalyzing the synthesis of esters under aqueous phase is an alternative with green and sustainable characteristics. Here, a biocatalyst esterase Bur01 was identified through genome sequencing and gene library construction from a Burkholderia ambifaria BJQ0010 with efficient ester synthesis property under aqueous phase for the first time. Bur01 was soluble expressed and the purified enzyme showed the highest activity at pH 4.0 and 40 °C. It had a broad substrate spectrum, especially for ethyl esters. The structure of Bur01 was categorized as a member of α/β fold hydrolase superfamily. The easier opening of lid under aqueous phase and the hydrophobicity of substrate channel contribute to easier access to the active center for substrate. Molecular docking and site-directed mutation demonstrated that the oxyanion hole Ala22, Met112 and π-bond stacking between His24 and Phe217 played essential roles in catalytic function. The mutants V149A, V149I, L159I and F137I enhanced enzyme activity to 1.42, 1.14, 1.32 and 2.19 folds due to reduced spatial resistance and increased hydrophobicity of channel and ethyl octanoate with the highest conversion ratio of 68.28 % was obtained for F137I. These results provided new ideas for developing green catalysts and catalytic basis of mechanistic studies for ester synthetase under aqueous phase.

Optimization of plasmid electrotransformation into Bacillus subtilis using an antibacterial peptide.

Bacillus subtilis can potentially serve as an efficient expression host for biotechnology due to its ability to secrete extracellular proteins and enzymes directly into the culture medium. One of the important challenges in the biotechnology industry is to optimize the transformation conditions of B. subtilis bacteria. This study aims to provide a new method to optimize the transformation conditions and improve the transformation efficiency of B. subtilis WB600. To increase the transformation efficiency in B. subtilis, two methods of adding CM11 antibacterial peptides to the bacterial medium along with electroporation and optimizing the variables including the growth medium composition, time to adding CM11 peptide, electroporation voltage, recovery medium, and cell recovery time are used. The results of this study showed that the addition of antimicrobial peptides (AMPs) with a concentration of 2 μg/ml increases the transformation efficiency by 4 times compared to the absence of AMP in the bacterial medium. Additionally, the findings from our study indicated that the most optimal rate of transformation for B. subtilis was observed at a voltage of 7.5 kV/cm, with a recovery period of 12 h. With the optimized method, the transformation efficiency came up to 1.69 × 104 CFU/µg DNA. This improvement in transformation efficiency will be attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for transformation of wild-type B. subtilis strains.

Characterizing Hereditary Hemolytic Anemias in a Hispanic Cohort Using Next Generation Sequencing

Background: Hereditary hemolytic anemias (HHA) are a heterogeneous group of nonimmune disorders characterized by increased red blood cell (RBC) destruction due to abnormalities of hemoglobin stability, defects of RBC metabolism, and disorders of RBC hydration. Although uncommon, they comprise important causes of anemia in children and adults by causing chronic or episodic hemolysis. Clinical, laboratory, and genetic heterogeneity characterize this group of disorders and make recognition challenging. The advent of next generation sequencing (NGS) has made accurate genetic diagnoses attainable, with an increasing number of studies reporting clinical utility in HHA. For patients with suspected HHA, multigene testing is available incorporating an NGS panel associated genes. However, there is limited data on the use of multigene NGS to characterize HHA in the Hispanic population. Herein, we describe results from NGS testing of a pilot cohort referred for HHA investigation to hematology clinics along the US/Mexico border. Methods: Pediatric and adult hematology clinics at Texas Tech University in El Paso, TX collaborated for testing on HHA referral and clinical case review. The diagnostic panel (AnemiaID/PerkinElmer/Revvity®) included 51 genes encoding RBC cytoskeletal proteins, membrane transporter, RBC enzymes, and certain bilirubin metabolism genes. The complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions were covered. Targeted gene capture and library construction for NGS were performed using a Whole Blood and Saliva kit and 150 base pair paired-end sequencing was done on Illumina® NGS systems at target average coverage of 80×. NGS output data were summarized descriptively for all patients and clinically correlated for diagnosis. Results: In this pilot cohort, 11 patients were tested during September 2022 to June 2023, age ranging 7-75 years, with females accounting for 64% of the group. Findings are reported in the Table below; Pathogenic variants leading to a definitive diagnosis were identified in 8 of 11 cases (73%). The most frequently mutated genes were SPTA1 followed by PIEZO1, SPTB, RHAG, GSR, SLC4A1 and TPI1. Approximately 58% of the mutations were reported as novel. Diagnoses included, most commonly, Hereditary Spherocytosis followed by Hereditary Pyropoikilocytosis and Congenital Dyserythropoietic Anemia. Compound heterozygosity leading to complex interactions was noted in 4 cases (36%), involving SPTA1 low expression alleles in trans (alpha-LELY and alpha-LEPRA) in 2 cases. Conclusion: Hereditary hemolytic anemia in the Hispanic population is characterized by complex molecular interactions and a high prevalence of novel mutations in RBC cytoskeleton/enzyme genes. NGS has clinical utility and should be considered for accurate diagnosis. Further research into the molecular basis and genetic spectrum of HHA in the Hispanic population is needed.

Construction of Human Immune and Naive scFv Phage Display Libraries.

Antibody phage display is a widely used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. In order to successfully generate antibodies using phage display, the basis is the construction of high-quality antibody gene libraries. Here, we describe detailed methods for the construction of such high-quality immune and naive scFv gene libraries of human origin. These protocols were used to develop human naive (e.g., HAL9/10) and immune libraries, which resulted in thousands of specific antibodies for all kinds of applications.

Antha-Guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries.

Protein engineering through directed evolutison is facilitated by the screening and characterization of protein libraries. Efficient and effective methods for multiple site-saturation mutagenesis, such as Darwin Assembly, can accelerate the sampling of relevant sequence space and the identification of variants with desired functionalities. Here, we present the automation of the Darwin Assembly method, using a Gilson PIPETMAX™ liquid handling platform under the control of the Antha software platform, which resulted in the accelerated construction of complex, multiplexed gene libraries error-free and with minimal hands-on time, while maintaining flexibility over experimental parameters through a graphical user interface rather than requiring user-driven library-dependent programming of the liquid handling platform. We also present an approach for barcoding libraries that overcomes amplicon length limitations in next generation sequencing and enables fast reconstruction of library reads.

Measuring the absolute abundance of the microbiome by adding yeast containing 16S rRNA gene from a hyperthermophile.

High‐throughput sequencing (HTS) of 16S rRNA gene amplicons provides compositional information regarding the microbial community, but not the absolute abundance of the bacteria. We aimed to develop a standardized method for quantifying the absolute abundance of bacteria in microbiome studies. To demonstrate the utility of our approach, we quantified the number of bacteria from the compositional data of the fecal and cecal microbiomes. The 16S rRNA gene of a hyperthermophile, Thermus aquaticus, was cloned into Pichia pastoris (yeast) genome, and an equivalent amount of the yeast was added to the stool and cecal samples of mice before DNA extraction. 16S rRNA gene library construction and HTS were performed after DNA extraction. The absolute abundances of bacteria were calculated using T. aquaticus reads. The average relative abundances of T. aquaticus in the five stool and five cecal samples were 0.95% and 0.33%, respectively, indicating that the number of bacteria in a cecum sample is 2.9 times higher than that in a stool sample. The method proposed for quantifying the absolute abundance of the bacterial population in this study is expected to overcome the limitation of showing only compositional data in most microbiome studies.

Microbial Diversity of Terrestrial Geothermal Springs in Armenia and Nagorno-Karabakh: A Review.

The microbial diversity of high-altitude geothermal springs has been recently assessed to explore their biotechnological potential. However, little is known regarding the microbiota of similar ecosystems located on the Armenian Highland. This review summarizes the known information on the microbiota of nine high-altitude mineralized geothermal springs (temperature range 25.8–70 °C and pH range 6.0–7.5) in Armenia and Nagorno-Karabakh. All these geothermal springs are at altitudes ranging from 960–2090 m above sea level and are located on the Alpide (Alpine–Himalayan) orogenic belt, a seismically active region. A mixed-cation mixed-anion composition, with total mineralization of 0.5 mg/L, has been identified for these thermal springs. The taxonomic diversity of hot spring microbiomes has been examined using culture-independent approaches, including denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene library construction, 454 pyrosequencing, and Illumina HiSeq. The bacterial phyla Proteobacteria, Bacteroidetes, Cyanobacteria, and Firmicutes are the predominant life forms in the studied springs. Archaea mainly include the phyla Euryarchaeota, Crenarchaeota, and Thaumarchaeota, and comprise less than 1% of the prokaryotic community. Comparison of microbial diversity in springs from Karvachar with that described for other terrestrial hot springs revealed that Proteobacteria, Bacteroidetes, Actinobacteria, and Deinococcus–Thermus are the common bacterial groups in terrestrial hot springs. Contemporaneously, specific bacterial and archaeal taxa were observed in different springs. Evaluation of the carbon, sulfur, and nitrogen metabolism in these hot spring communities has revealed diversity in terms of metabolic activity. Temperature seems to be an important factor in shaping the microbial communities of these springs. Overall, the diversity and richness of the microbiota are negatively affected by increasing temperature. Other abiotic factors, including pH, mineralization, and geological history, also impact the structure and function of the microbial community. More than 130 bacterial and archaeal strains (Bacillus, Geobacillus, Parageobacillus, Anoxybacillus, Paenibacillus, Brevibacillus Aeribacillus, Ureibacillus, Thermoactinomyces, Sporosarcina, Thermus, Rhodobacter, Thiospirillum, Thiocapsa, Rhodopseudomonas, Methylocaldum, Desulfomicrobium, Desulfovibrio, Treponema, Arcobacter, Nitropspira, and Methanoculleus) have been reported, some of which may be representative of novel species (sharing 91–97% sequence identity with their closest matches in GenBank) and producers of thermozymes and biomolecules with potential biotechnological applications. Whole-genome shotgun sequencing of T. scotoductus K1, as well as of the potentially new Treponema sp. J25 and Anoxybacillus sp. K1, were performed. Most of the phyla identified by 16S rRNA were also identified using metagenomic approaches. Detailed characterization of thermophilic isolates indicate the potential of the studied springs as a source of biotechnologically valuable microbes and biomolecules.

Screening of Mutations Related to Hereditary Anemias Using NGS-Targeted Sequencing Panel

Hereditary forms of anemia arise due to defects in erythrocyte differentiation from bone marrow precursor cells, or shortened survival of erythrocytes. Both cases result in the deficiency of mature cells in the bloodstream. Defects in the erythrocyte membrane proteins (e.g. hereditary spherocytosis and elliptocytosis/ pyropoikilocytosis) are common, and they manifest in association with morphological changes in the red blood cells. Other causes of hemolytic anemias include hemoglobinopathies (e.g. sickle cell diseases, thalassemias, etc) and enzymopathies (e.g. Glucose 6 phosphate dehydrogenase deficiency and pyruvate kinase deficiency), caused by defects in metabolic enzymes. Hereditary anemia can also be caused by erythroid lineage disorders such as congenital dyserythropoietic anemias (CDA), characterized by distinct morphologic abnormalities of marrow erythroblasts. Diseases that result in hereditary anemias are difficult to diagnose, due to the large clinical and genetic heterogeneity. The use of conventional techniques for the systematic screening of all potentially implicated genes would be impractical, costly and time-consuming. To address this issue, we have customized a rapid comprehensive next-generation sequencing-based assay that evaluates 58 genes with published disease-causing mutations for future clinical application. Of these genes, 27 were related with RBC cytoskeletal disorders and enzymopathies, 8 were involved with CDAs and 23 genes were related with hemochromatosis, sideroblastic anemia and porphyria. Our panel covers the complete coding region, splice site junctions, and some promoter regions. Exon capture probes were designed with the aid of the Sure Design program (Agilent Technologies, Inc., CA, USA). Targeted gene capture and library construction for next-generation sequencing (NGS) were performed using Haloplex kit (Agilent Technologies, Santa Clara, USA). Enriched samples were then sequenced on an Illumina HiSeq 2500 sequencer with 150 base pair, paired-end reads. Overall, the quality scores obtained were high, with an average of 91% of the bases analyzed with >Q30. This means that the probability that a given base is wrong is less than 0.1%. Sequencing reads were aligned to the human genome reference sequence and analysis of coverage and variants was completed using Sure Call software (Agilent Technologies, Inc., CA, USA). The validation included the benchmark sample NA12878 (Coriell Institute, NJ, USA) in triplicate. All variants were detected using Haloplex amplification followed by NGS were compared to the results available in the coding region sequencing databases of the NA12878 sample. The panel showed a sensitivity of 98.7% and a specificity of 99.9%. We also tested our panel in 43 patients with hereditary anemia searching for causative variants. Possible pathogenic variants were identified in 37 patients (86%) and explained the etiology of the diseases in 69.8% of the cases. Variants in the β-spectrin (SPTB gene) were the most frequent cause of hereditary spherocytosis (HS) in our sample, identified in 13 out of the 26 patients with previous diagnosis of HS (50%). Variants in the ankyrin (ANK1) were the second most frequent cause of HS, identified in 6 cases. Variants in SPTA1, SLC4A1, PKLR, CDAN1, G6PD, HFE and KIF23 genes were also identified. Most of the identified variants were novel. To conclude, next-generation sequencing provides a cost-effective and relatively rapid approach to molecular characterization of a disease. The identification of pathogenic variants is essential for genetic counselling, pre-implantation genetic diagnosis and even for future gene therapy. The panel we developed proved to be robust, with very low false positive and negative results and successfully determined the molecular causes of hereditary anemia in most cases. DisclosuresNo relevant conflicts of interest to declare.

Microbiomic profiles of bile in patients with benign and malignant pancreaticobiliary disease.

417 Background: In pancreaticobiliary (PB) cancers, there is a paucity of data on predictive and pathophysiologic role of the biliary microbiome. We analyzed bile collected from patients with benign and malignant PB diseases to identify microbiomic signatures associated with malignancy. Methods: We collected bile samples from consenting patients during routine endoscopic retrograde cholangiopancreatography at the Cleveland Clinic, approved by the Institutional Review Board. DNA was extracted from bile specimens using PowerViral RNA/DNA Isolation kit. Bacterial 16S rRNA gene amplification and library construction were performed according to the 16S Metagenomic Sequencing Library Preparation guide from Illumina. Post-sequencing analysis was done using QIIME (Quantitative Insights Into Microbial Ecology), Bioconductor phyloseq, microbiomeSeq and mixMC packages. Results: Of 46 enrolled patients, 32 had PB cancers including pancreatic (N = 25), cholangiocarcinoma (N = 6), and gallbladder (N = 1). The rest (N = 14) had benign PB diseases including acute and chronic pancreatitis, and gallstones. Using multivariate approach in mixMC to classify Operational Taxonomic Units (OTUs), we found a predominance of genera Dicekeya (p = 0.0002), [ Eubacterium] hallii group (p = 0.0007) , Bacteroides (p = 0.00099) , Faecalibacterium (p = 0.007) , Facklamia (p = 0.013) , Peptococcus (p = 0.013) , Bergeyella (p = 0.0024) , Lachnospira (p = 0.026) , and Lactobacillus (p = 0.025) in bile samples from PB cancers as compared to benign PB diseases. Furthermore, bile samples from patients with pancreatic cancer showed an increased abundance of genera Enterobacter, Parabacteroides, Atopobium, Alloprevotella, Prevotella 7, Acinetobacter, Bergeyella, Clostridium sensu stricto, Lactobacillus, and Rothia; and a decreased abundance of genera Tannerella, Peptococcus, Colinsella, Capnocytophaga, Achromobacter, Ruminococcus 2, Bacteroides, Alistipes, Barnesiella,, Lachnoclostridium, Lautropia, Akkermansia, and Christensenellaceae R-7 group as compared to bile samples from patients with cholangiocarcinoma. Conclusions: Distinct microbiome signatures are associated with benign and malignant PB diseases. There is a difference in the relative abundance of OTUs in bile samples between patients with benign PB diseases vs PB cancers, and between pancreatic cancer vs cholangiocarcinoma. Our findings raise the possibility that either these OTUs have a role in carcinogenesis, or that changes in the microenvironment of benign PB diseases differ from PB cancers leading to distinct separation of the OTU clusters. Further studies to explore and validate our findings are needed.

In Memoriam: Marvin A. Van Dilla: 1919-2019.

© 2020 International Society for Advancement of Cytometry.

Discovery and development of a novel short-chain fatty acid ester synthetic biocatalyst under aqueous phase from Monascus purpureus isolated from Baijiu

Short-chain fatty acid esters are important flavor chemicals in Chinese traditional fermented Baijiu. Monascus purpureus was recognized as an important microorganism contributing to ester synthesis. However, the molecular basis for ester synthesis was still lacking. The present work combined genome sequencing, transcriptome sequencing, gene library construction, and enzyme engineering to discover a novel catalyst from M. purpureus (isolated from Baijiu fermentation starter). Enzyme LIP05, belonging to the α/β hydrolase family, was identified to synthesize short-chain fatty acid esters under aqueous phase. After deleting the lid domain of LIP05, the synthesis of ethyl pentanoate, ethyl hexanoate, ethyl octanoate, or ethyl decanoate was achieved. Ethyl octanoate with the highest conversion ratio of 93.7% was obtained with the assistance of ultrasound. The study reveals the molecular basis for synthesizing short-chain fatty acid esters by M. purpureus and will promote the application of the species or the enzyme in food industry.

Determination of Pathogenicity of Breast Cancer 1 Gene Variants using the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Guidelines.

ObjectivesMolecular diagnostic laboratories screen for mutations in disease-causing genes in order to confirm a clinical diagnosis. The classification of DNA variants as ‘pathogenic’ or ‘likely pathogenic’ mutations creates a workflow bottleneck, which becomes increasingly challenging as greater number of genes are screened. The classification challenge is also acute if there are conflicting reports regarding pathogenicity and differing classification criteria between laboratories. This study aimed to compare two procedures for the classification of variants in the breast cancer (BRCA)1 gene.MethodsThis bioinformatic study was conducted at LabPLUS, Auckland, New Zealand, from February to June 2017. DNA was extracted from peripheral blood samples of 30 patients and gene library construction was carried out using a commercially available targeted panel for the BRCA1 and BRCA2 genes. The genes were subsequently sequenced and the sequence data analysed. The guidelines published by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) provides a comprehensive framework for the interpretation of variants in genes that are associated with Mendelian disorders. The use of these guidelines were compared to the variant classifications that were achieved by reference to those reported in the BRCA Exchange database.ResultsThe results showed concordance between the two classification protocols for a panel of 30 BRCA1 gene variants, although the transparency in following the ACMG/AMP guidelines provides a diagnostic laboratory with a generalisable approach that allows laboratory-directed revisions to be undertaken in light of new information.ConclusionThe ACMG/AMP-based guidelines were applied to a cohort of patients with BRCA1 gene variants. The use of these guidelines provides a system which creates consistency in variant interpretation and supports subsequent clinical management.

Microbiome signature of bile from pancreatic and biliary tract cancer patients: A pilot study.

e15744 Background: Recent studies especially in murine models have linked meta-organismal pathways in the gut microbial community to cancers of the pancreas and the biliary tract. However, data on microbiome in the biliary pool in patients to establish models for oncogenesis in the pancreatobiliary (PB) habitat have been limited. We analyzed bile collected from a pilot series of patients to identify microbiome signatures associated with malignancy. Methods: We collected bile samples from patients during routine endoscopic retrograde cholangiopancreatography in this study approved by the Cleveland Clinic Institutional Review Board for Human Subjects’ Protection. Of 10 patients, there were 5 with pancreatic ductal adenocarcinoma (PDA), 3 with cholangiocarcinoma (CC), 1 with ampullary adenocarcinoma, and 1 with gallstone pancreatitis. DNA was extracted from bile specimens using PowerViral RNA/DNA Isolation kit. Bacterial 16S rRNA gene amplification and library construction were performed according to the 16S Metagenomic Sequencing Library Preparation guide from Illumina. Post-sequencing analysis was done using QIIME (Quantitative Insights Into Microbial Ecology) and MICCA (MICrobial Community Analysis). Results: Most reads were from phyla Firmicutes (57.9%) and Proteobacteria (14.9%). One benign specimen (pancreatitis) separated clearly from the rest showing 98.9% of reads from Clostridium sensu stricto (phylum Firmicutes). Analysis of beta diversity showed six samples clustering tightly. Of these, 5 were PDA and 1 was CC. A second cluster included remaining 3 samples, 2 with CC and 1 with PDA; this latter had higher abundance of phyla Fusobacteria (90.6%) and Verrucomicrobia (92.9%). Conclusions: Select bacteria are differentially increased in malignant and benign PB diseases. Furthermore, distinct microbiome signatures may be associated with cancer in the pancreatic and biliary habitats. We intend to evaluate these findings in larger sample sizes and determine associations with clinical outcomes and response to treatment.

Use of Next Generation Sequencing Panel for Routine Diagnosis of Hereditary Hemolytic Anemias

Hereditary hemolytic anemia (HHA) are a heterogeneous group of disorders due to germline mutations of the red cell cytoskeleton (e.g. hereditary spherocytosis (HS) and hereditary elliptocytosis/pyropoikilocytosis (HE/HPP)) or enzyme deficiencies (e.g. glucose 6 phosphate dehydrogenase deficiency (G6PD) and pyruvate kinase deficiency (PKD). Routine morphological and biochemical analysis may be inconclusive in neonates due to the physiological nature of erythroid cell maturation and can also be misleading in transfusion-dependent patients. Additionally, there has been increasing awareness of inherited red cell membrane disorders that are not easily identified by routine laboratory approaches. For example, clinically insignificant defects of RBC membrane genes (e.g. alpha LELY and alpha LEPRA in SPTA1), which can be present in the parents without significant hemolysis, may result in compound heterozygosity in the offspring, causing severe morbidity or even mortality due to significant hemolysis. Awareness of these low expression alleles is important for genetic counseling purposes. Molecular studies, although becoming more mainstream, have not been used extensively to diagnose these disorders. This is most likely due to the complex genetic nature of these disorders (e.g. large genes with multiple exons involved, and multi-gene disorders (i.e. hyperbilirubinemia due to HS as well as involvement of genes involved in bilirubin metabolism). The accessibility of next generation sequencing (NGS) methods in the clinical laboratory has made diagnosing complex genetic disorders feasible.Our current diagnostic panel includes 28 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for NGS are performed using a Sure Select kit (Agilent). Indexed samples are quantified using qPCR and then pooled prior to sequencing on the Illumina NextSeq or HiSeq instruments. Samples are sequenced using 150 bp paired-end sequencing. This panel includes genes responsible for RBC membrane defects, enzyme deficiencies, as well as bilirubin uridine diphosphate glucuronosyltransferase (UGT1A) genes that have a distinct role in hyperbilirubinemia.We now report the first 268 patients evaluated using our NGS panel between 2015-2018. These patients were evaluated using an Institutional Review Board Protocol (IRB - 00077285). The age of the patients ranged from newborn to 68 years. These patients presented with symptoms ranging from mild lifelong anemia to severe hemolytic anemia with extreme hyperbilirubinemia. Genetic variants were classified according to the American College of Medical Genetics (ACMG) guidelines.We identified pathogenic and likely pathogenic variants in 64/268 (24%) patients that were clearly responsible for the disease phenotype (e.g. moderate to severe hemolytic anemia). Approximately half of them were novel mutations. Moreover, 29/268 (11%) of patients were homozygous for a promoter polymorphism in the UGT1A1 gene A(TA)7TAA (UGT1A1*28), which may lead to reduced expression of the UGT1A1 gene and Gilbert's syndrome. Furthermore, 4/29 UGT1A1 polymorphism cases were associated with pathogenic spectrin mutations, likely increasing the severity of the clinical phenotype in these patients. Overall, the most commonly mutated genes were SPTB and SPTA1, encoding spectrin subunits, followed by PKLR and ANK1 (Table 1). Complex interactions between variants in the SPTA1 gene and the common alpha-LELY and alpha-LEPRA alleles were predicted to be associated with HPP and autosomal recessive HS in 12/64 patients. Furthermore, 23/268 (9%) patients had mutations that were predicted to cause moderate to severe anemia if inherited with another mutation, making them important for genetic counseling purposes (data not shown). Our results demonstrate that many patients with hemolytic anemia harbor complex combinations of known and novel mutations in RBC cytoskeleton/enzyme genes. Many variants of unknown significance were also identified that could potentially contribute to disease.To conclude, the use of NGS provides a cost-effective and comprehensive method to assist in the diagnosis of hemolytic anemias, especially in instances where complex gene-gene interactions are suspected. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Construction of Human Immune and Naive scFv Libraries.

Antibody phage display is the most commonly used in vitro selection technology for the generation of human recombinant antibodies and has yielded thousands of useful antibodies for research, diagnostics, and therapy. The prerequisite for successful generation of antibodies using phage display is the construction of high-quality antibody gene libraries. Here, we give the detailed methods for the construction of human immune and naive scFv gene libraries.

Chemoselective Coupling Preserves the Substrate Integrity of Surface-Immobilized Oligonucleotides for Emulsion PCR-Based Gene Library Construction.

Combinatorial bead libraries figure prominently in next-generation sequencing and are also important tools for in vitro evolution. The most common methodology for generating such bead libraries, emulsion PCR (emPCR), enzymatically extends bead-immobilized oligonucleotide PCR primers in emulsion droplets containing a single progenitor library member. Primers are almost always immobilized on beads via noncovalent biotin-streptavidin binding. Here, we describe covalent bead functionalization with primers (∼106 primers/2.8-μm-diameter bead) via either azide-alkyne click chemistry or Michael addition. The primers are viable polymerase substrates (4-7% bead-immobilized enzymatic extension product yield from one thermal cycle). Carbodiimide-activated carboxylic acid beads only react with oligonucleotides under conditions that promote nonspecific interactions (low salt, low pH, no detergent), comparably immobilizing primers on beads, but yielding no detectable enzymatic extension product. Click-functionalized beads perform satisfactorily in emPCR of a site-saturation mutagenesis library, generating monoclonal templated beads (104-105 copies/bead, 1.4-kb amplicons). This simpler, chemical approach to primer immobilization may spur more economical library preparation for high-throughput sequencing and enable more complex surface elaboration for in vitro evolution.

Using a Next Generation Sequencing Panel to Discover the Obscure Causes of Hereditary Hemolytic Anemias

Hereditary hemolytic anemias are a heterogeneous group of disorders with consequences ranging from non-anemic hemolysis to severe life-threatening anemia. However, the late morbidity in patients without transfusions is often underappreciated because of erythropoietic compensatory stimulation inducing hematopoiesis by erythroferrone/hepcidin axis. Principal causes of hereditary hemolytic anemias are germline mutations of red cell cytoskeleton (e.g. hereditary spherocytosis and elliptocytosis/pyropoikilocytosis) or enzyme deficiencies (e.g. Glucose 6 phosphate dehydrogenase deficiency and pyruvate kinase deficiency). Routine morphological and biochemical analysis may be inconclusive and misleading particularly in transfusion-dependent infants and children. Molecular studies have not been extensively used to diagnose these disorders due to the complex genetic nature of these disorders, and multi-gene disorders. In these cases, patients may undergo multiple rounds of single gene testing, which can be very costly and time consuming. The advent of next generation sequencing (NGS) methods in the clinical laboratory has made diagnosing complex genetic disorders feasible. Our diagnostic panel includes 28 genes encoding cytoskeletal proteins and enzymes, and covers the complete coding region, splice site junctions, and, where appropriate, deep intronic or regulatory regions. Targeted gene capture and library construction for next-generation sequencing (NGS) was performed using Sure Select kit (Agilent Technologies, Santa Clara, USA). Prior to sequencing on the Illumina Next Seq, (Illumina Inc) instrument, indexed samples are quantified using qPCR and then pooled. Samples were sequenced using 2x150 paired end sequencing. We now report the first 68 patients evaluated using our NGS panel. The age of the patients ranged from newborn to 62 years. These patients presented with symptoms ranging from mild lifelong anemia to severe hemolytic anemia with extreme hyperbilirubinemia. Genetic variants were classified using the American College of Medical Genetics (ACMG) guidelines. We identified pathogenic variants in 11 patients and likely pathogenic variants in 12 others, the majority of these were novel. Many variants with unknown significance were also identified that could potentially contribute to disease. The most commonly mutated genes were SPTB and SPTA1, encoding spectrin subunits. Some complex interactions were uncovered i.e. SPTA1 mutations along with alpha LELY leading to hereditary pyropoikilocytosis; Spectrin variants along with Gilbert syndrome causing severe hyperbilirubinemia in neonates; and Spectrin variants in combination with PKLR and G6PD variants. Our results demonstrate that many patients with hemolytic anemia harbor complex combinations of known and novel mutations in RBC cytoskeleton/enzyme genes, but their clinical significance is further augmented by polymorphisms of UGT1A1 gene contributing to severe neonatal hyperbilirubinemia and its consequences. To conclude, next-generation sequencing provides a cost-effective and relatively rapid approach to molecular diagnosis, especially in instances where traditional testing failed. We have used this technology successfully to determine the molecular causes of hemolytic anemia in many cases with no prior family history. DisclosuresYaish:Octapharma: Other: Study investigator.

Bacterial communities associated with invasive populations of Bactrocera dorsalis (Diptera: Tephritidae) in China.

The oriental fruit fly Bactrocera dorsalis (Hendel) is a destructive insect pest of a wide range of fruits and vegetables. This pest is an invasive species and is currently distributed in some provinces of China. To recover the symbiotic bacteria of B. dorsalis from different invasion regions in China, we researched the bacterial diversity of this fruit fly among one laboratory colony (Guangdong, China) and 15 wild populations (14 sites in China and one site in Thailand) using DNA-based approaches. The construction of 16S rRNA gene libraries allowed the identification of 24 operational taxonomic units of associated bacteria at the 3% distance level, and these were affiliated with 3 phyla, 5 families, and 13 genera. The higher bacterial diversity was recovered in wild populations compared with the laboratory colony and in samples from early term invasion regions compared with samples from late term invasion regions. Moreover, Klebsiella pneumoniae and Providencia sp. were two of the most frequently recovered bacteria, present in flies collected from three different regions in China where B. dorsalis is invasive. This study for the first time provides a systemic investigation of the symbiotic bacteria of B. dorsalis from different invasion regions in China.