scholarly journals Antibody Upstream Sequence Diversity and Its Biological Implications Revealed by Repertoire Sequencing

2020 ◽  
Author(s):  
Yan Zhu ◽  
Xiujia Yang ◽  
Jiaqi Wu ◽  
Haipei Tang ◽  
Qilong Wang ◽  
...  
Keyword(s):  
Cynomolgus Macaque ◽  
Pcr Amplification ◽  
Mrna Translation ◽  
Variable Region ◽  
Antibody Engineering ◽  
Upstream Sequence ◽  
Nucleotide Polymorphisms ◽  
Bioinformatics Pipeline ◽  
Repertoire Sequencing ◽  
Rapid Amplification

AbstractThe sequence upstream of antibody variable region (Antibody Upstream Sequence, or AUS) consists of 5’ untranslated region (5’ UTR) and two leader regions, L-PART1 and L-PART2. The sequence variations in AUS affect the efficiency of PCR amplification, mRNA translation, and subsequent PCR-based antibody quantification as well as antibody engineering. Despite their importance, the diversity of AUSs has long been neglected. Utilizing the rapid amplification of cDNA ends (5’RACE) and high-throughput antibody repertoire sequencing (Rep-Seq) technique, we acquired full-length AUSs for human, rhesus macaque (RM), cynomolgus macaque (CM), mouse, and rat. We designed a bioinformatics pipeline and discovered 2,957 unique AUSs, corresponding to 2,786 and 1,159 unique sequences for 5’ UTR and leader, respectively. Comparing with the leader records in the international ImMunoGeneTics (IMGT), while 529 were identical, 313 were with single nucleotide polymorphisms (SNPs), 280 were totally new, and 37 updated the incomplete records. The diversity of AUSs’ impact on related antibody biology was also probed. Taken together, our findings would facilitate Rep-Seq primer design for capturing antibodies comprehensively and efficiently as well as provide a valuable resource for antibody engineering and the studies of antibody at the molecular level.

scholarly journals NanoHIV: A Bioinformatics Pipeline for Producing Accurate, Near Full-Length HIV Proviral Genomes Sequenced Using the Oxford Nanopore Technology

Cells ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2577
Author(s):  
Imogen A. Wright ◽  
Kayla E. Delaney ◽  
Mary Grace K. Katusiime ◽  
Johannes C. Botha ◽  
Susan Engelbrecht ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Cost Effective ◽  
Full Length ◽  
Sequencing Error ◽  
Consensus Building ◽  
Genome Sequences ◽  
Bioinformatics Pipeline ◽  
Oxford Nanopore ◽  
Single Genome ◽  
Hiv 1

HIV-1 proviral single-genome sequencing by limiting-dilution polymerase chain reaction (PCR) amplification is important for differentiating the sequence-intact from defective proviruses that persist during antiretroviral therapy (ART). Intact proviruses may rebound if ART is interrupted and are the barrier to an HIV cure. Oxford Nanopore Technologies (ONT) sequencing offers a promising, cost-effective approach to the sequencing of long amplicons such as near full-length HIV-1 proviruses, but the high diversity of HIV-1 and the ONT sequencing error render analysis of the generated data difficult. NanoHIV is a new tool that uses an iterative consensus generation approach to construct accurate, near full-length HIV-1 proviral single-genome sequences from ONT data. To validate the approach, single-genome sequences generated using NanoHIV consensus building were compared to Illumina® consensus building of the same nine single-genome near full-length amplicons and an average agreement of 99.4% was found between the two sequencing approaches.

scholarly journals Evaluation of SSR and SNP markers in Rubus glaucus Benth progenitors selection

2019 ◽  
Vol 41 (1) ◽  
Author(s):  
Ana María López ◽  
Carlos Felipe Barrera ◽  
Marta Leonor Marulanda
Keyword(s):  
Plant Breeding ◽  
Technological Development ◽  
Pcr Amplification ◽  
Snp Markers ◽  
Morphological Features ◽  
Agricultural Product ◽  
Nucleotide Polymorphisms ◽  
Family Selection ◽  
Planting Material ◽  
Small Producers

Abstract Rubus glaucus Benth (known as “mora de castilla”) is a Colombian agricultural product, with probably, the major potential. This fruit combines features of Idaeobatus and Rubus subgenera. Despite its recognized importance in the economy of small producers, this crop has received little technological development; as a result, sowing procedures of this specie is done by using local cultivars asexually propagated by producers. Associated Rubus producers in Colombia has noticed the necessity to formalize the offer of planting material, starting with plant breeding programs tending to the obtention of more productive varieties with morphological features that facilitates cultural activities. This study presents the results of the evaluation of SSR and SNPs markers obtained in previous experimental works from a RNA-Seq transcriptome analysis. It was evaluated 15 promissory R. glaucus cultivars, which could be potential progenitors in future breeding schemes. Genetic characterization was accomplished by testing 22 SSR microsatellite and 78 single nucleotide polymorphisms (SNPs) markers. From evaluated SSR markers, 15 yielded positive PCR amplification generating 29 loci and 58 alleles. From evaluated SNPs markers, 36 yielded positive PCR amplification. Obtained sequences from amplified products with SNPs showed high homology with species belonging to Rosaceae family. Selection criteria of progenitors were based on the results of molecular characterizations and useful morphological features in the culture management. This research demonstrates the utility of molecular markers to assess genetic diversity of potential progenitors susceptible to plant breeding processes. This process, highly known as development of parental populations, determines in a great manner the success of plant breeding processes.

scholarly journals Identification of MC4R gene and its association with body weight and body size in Kebumen Ongole Grade cattle

2018 ◽  
Vol 43 (2) ◽  
pp. 87
Author(s):  
D. Maharani ◽  
A. Fathoni ◽  
S. Sumadi ◽  
T. Hartatik ◽  
M. Khusnudin
Keyword(s):  
Body Length ◽  
Growth Traits ◽  
Average Daily Gain ◽  
Pcr Amplification ◽  
Growth Trait ◽  
Chest Circumference ◽  
Nucleotide Polymorphisms ◽  
Chi Square ◽  
Mc4r Gene ◽  
Genotype Frequencies

MC4R gene is known as an important candidate gene for the growth trait. The purpose of this research was to identify the MC4R gene in Kebumen Ongole grade cattle and examine its association with growth traits. Data of birth weight (BW), weaning weight (WW), birth body length (BBL), birth chest circumference (BCC), birth shoulder height (BSH), weaning body length (WBL), weaning chest circumference(WCC), weaning shoulder height (WSH) and average daily gain (ADG) were collected and used for analysis of MC4R gene. Sixty blood samples were collected for DNA isolation and PCR amplification. The single nucleotide polymorphisms (SNP) g.1133 C>G was used for genotyping by using PCR-RFLP methods. The frequenciy of G allele (0.59) was greater than C allele (0.41). The highest genotype frequencies have been detected in CG heterozygote animals (0.52) followed by GG (0.33) and CC (0.15) in homozygote animals. The results of Pearson ‘s Chi-square test indicated that the population was not deviate (P>0.05) from the Hardy-Weinberg equilibrium (HWE). The SNP g. 1133 C>G of MC4R gene indicated affecting high birth body length with GG genotype (P<0.05). In conclusion, the SNP g. 1133 C>G may can be a marker for birth body length of calf selection.

scholarly journals Detection and Quantification of Influenza Virus Defective Viral Genomes from NGS Datasets Obtained after RT or RT-PCR Product Sequencing

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 84
Author(s):  
Jeremy Boussier ◽  
Sandie Munier ◽  
Bernadette Crescenzo-Chaigne ◽  
Sylvie Behillil ◽  
Vincent Enouf ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Molecular Mechanisms ◽  
Pcr Amplification ◽  
Influenza Viruses ◽  
Detection Methods ◽  
Rt Pcr ◽  
Bioinformatics Pipeline ◽  
Genomic Rnas ◽  
Viral Genomes ◽  
Detection And Quantification

Like most RNA viruses, influenza viruses (IAV) generate defective viral genomes (DVGs) during viral replication. Although there is accumulating evidence of a biological impact of DVGs, the molecular mechanisms leading to their production remain to be unveiled. Various next-generation sequencing (NGS) technologies and detection methods can be used to characterize DVGs. Here, we developed a bioinformatics pipeline called DG-seq to quickly identify and quantify DVGs in influenza viral stocks and compared two processing methods for NGS, with or without PCR amplification. To evaluate the performance of the DG-seq pipeline, we used either synthetic in-vitro-transcribed DVGs mixed with the full set of synthetic full-length genomic RNAs, or biological RNA samples extracted in duplicate from three IAV stocks: mutant viruses with a K635A or a R638A mutation in the PA subunit of the polymerase that impairs viral transcription, and their wild-type (WT) counterpart. Viral genomic RNAs were reverse-transcribed and either directly subjected to Illumina sequencing (RT-seq) or PCR-amplified prior to sequencing (RT-PCR-seq). Both methods displayed a good reproducibility between batches, with a lower detection rate but a more accurate quantification of DVGs in RT-seq samples. The PA mutants produced more DVGs than the WT virus, derived mostly from the polymerase gene segments, but also from the NA and HA segments, suggesting that an imbalance between transcription and replication can promote DVG production. Breakpoints occurred near the segment extremities, with no hotspot identified. Interestingly, we observed short direct A/T-rich repeats adjacent to the breakpoint ends at a significantly higher frequency than in the random case. This work provides the first comparison of DVG detection and quantification from NGS data obtained in the presence or absence of PCR amplification and gives novel insight into the mechanisms of influenza virus DVG production.

scholarly journals Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients

2020 ◽  
Vol 21 (10) ◽  
pp. 3555
Author(s):  
Abdul Qawee Mahyoob Rani ◽  
Tetsushi Yamamoto ◽  
Tatsuya Kawaguchi ◽  
Kazuhiro Maeta ◽  
Hiroyuki Awano ◽  
...  
Keyword(s):  
Pcr Amplification ◽  
Alternative Polyadenylation ◽  
Coding Region ◽  
Terminal Exon ◽  
Potential Implication ◽  
Human Genes ◽  
Cardiac Muscles ◽  
Rapid Amplification ◽  
Dmd Gene ◽  
Ecg Abnormalities

The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3′ terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3′-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5′ end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.

scholarly journals High Genetic Variability of the agr Locus in Staphylococcus Species

2002 ◽  
Vol 184 (4) ◽  
pp. 1180-1186 ◽  
Author(s):  
Philippe Dufour ◽  
Sophie Jarraud ◽  
Francois Vandenesch ◽  
Timothy Greenland ◽  
Richard P. Novick ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Quorum Sensing ◽  
Sequence Divergence ◽  
Pcr Amplification ◽  
Variable Region ◽  
Cysteine Residue ◽  
Multiple Alignment ◽  
Open Reading Frames ◽  
Signal Transduction System ◽  
Reading Frames

ABSTRACT The agr quorum-sensing and signal transduction system was initially described in Staphylococcus aureus, where four distinct allelic variants have been sequenced. Western blotting suggests the presence of homologous loci in many other staphylococci, and this has been confirmed for S. epidermidis and S. lugdunensis. In this study we isolated agr-like loci from a range of staphylococci by using PCR amplification from primers common to the six published agr sequences and bracketing the most variable region, associated with quorum-sensing specificity. Positive amplifications were obtained from 14 of 34 staphylococcal species or subspecies tested. Sequences of the amplicons identified 24 distinct variants which exhibited extensive sequence divergence with only 10% of the nucleotides absolutely conserved on multiple alignment. This variability involved all three open reading frames involved in quorum sensing and signal transduction. However, these variants retained several protein signatures, including the conserved cysteine residue of the autoinducing peptide, with the exception of S. intermedius of pigeon origin, which contained a serine in place of cysteine at this position. We discuss hypotheses on the mode of action and the molecular evolution of the agr locus based on comparisons between the newly determined sequences.

Use of Pharmacogenetics and Clinical Factors To Predict the Maintenance Dose of Warfarin.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 550-550
Author(s):  
B. F. Gage ◽  
C. S. Eby ◽  
J. A. Johnson ◽  
M. J. Rieder ◽  
P. M. Ridker ◽  
...  
Keyword(s):  
African American ◽  
Warfarin Therapy ◽  
Validation Cohort ◽  
Pcr Amplification ◽  
Warfarin Dose ◽  
Regression Equation ◽  
Nucleotide Polymorphisms ◽  
Clinical Factors ◽  
Derivation Cohort ◽  
Unit Increase

Abstract Background Initiation of warfarin therapy using trial-and-error dosing can cause bleeding. Clinical factors explain only 20%–30% of the variability in the therapeutic dose of warfarin. Single nucleotide polymorphisms (SNPs) in the cytochrome P450 2C9 (CYP2C9) gene correlate with the clearance of S-warfarin and SNPs in the vitamin K epoxide reductase (VKORC1) gene predict warfarin sensitivity. We test the hypothesis that the combination of clinical and pharmacogenetic information can predict the therapeutic warfarin dose. Methods We collected DNA, demographic variables, laboratory values, and medication histories from patients taking warfarin. Subjects either attended an outpatient anticoagulation clinic or participated in the PREVENT (prevention of venous thromboembolism) study. After PCR amplification, we used Pyrosequencing® to genotype DNA regions for 2 coding CYP2C9 SNPs, *2 (C430T) and *3 (A1075C), and for 4 noncoding VKORC1 SNPs: C861A, A5808C, G6853C, and G9041A. Using multiple regression, we quantified the association between therapeutic warfarin dose and clinical and genetic factors in a derivation cohort of 900 participants and a validation cohort of 100 participants. Results The VKORC1 G6853C SNP was the first variable to enter the stepwise regression equation and was associated with a 27% decrease in the warfarin dose per allele in Caucasian patients. The VKORC1 A5808C SNP was associated with a 33% decrease per allele in warfarin dose in African-American patients. Other significant (p &lt; 0.05) predictors of the therapeutic warfarin dose, in order of entry into the regression equation and their effect on warfarin dose were: body surface area (+12% per SD increase), CYP2C9*3 (−33% per allele), CYP2C9*2 (−20% per allele), age (−7% per decade), target INR (+8% per 0.5 unit increase), amiodarone use (−24%), African-American race (+12%), smoker (+9%), and simvastatin or fluvastatin use (−5%). A dosing equation that included these pharmacogenetic and clinical factors explained 52% of the dose variability in derivation cohort and 55% of the variability in the validation cohort. Conclusions The therapeutic warfarin dose can be estimated from clinical and pharmacogenetic factors that can be obtained when warfarin is started. Use of this dosing equation has potential to aid in the prediction of an optimal warfarin dose, which may decrease the risk of bleeding during the initiation of warfarin therapy.

Analysis of Immunogenetic Factors in Paroxysmal Nocturnal Hemoglobinuria Reveal Increased Frequency of IL-10 Low Secretor Genotype and Correlates with Induced IL-10 Production.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 983-983
Author(s):  
Giridharan Ramsingh ◽  
Zachary Nearman ◽  
Chris Hung ◽  
Serio Bianca ◽  
Antonio Risitano ◽  
...  
Keyword(s):  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Direct Sequencing ◽  
Pcr Amplification ◽  
Hla Typing ◽  
Nucleotide Polymorphisms ◽  
Specific Pcr ◽  
Hla Ligand ◽  
Functional Consequences ◽  
Immunologic Diseases ◽  
Common Pathogenesis

Abstract Aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) have a common pathogenesis related to an immune attack on hematopoietic progenitor and stem cells. While the inciting events are not known, it is possible that complex immunogenetic predisposition factors exist which, in the context of exogenous influences, determine risk for these diseases. They include HLA background, KIR genotype, cytokine and immunomodulatory gene polymorphisms as well as gene variants involved in peptide processing and presentation. We have empirically selected a number of important polymorphisms that were described in the context of various immunologic diseases and studied their frequency in a large cohort (N=57) of PNH patients. As binding of KIR to the appropriate HLA ligand (KIR-L) can modulate activation of Nk-cells and cytotoxic T lymphocytes, we examined the combined impact of KIR/KIR-L genotypes on the risk of PNH and PNH/AA syndrome. PNH showed a decreased frequency of inhibitory KIR-2DL1 and KIR-2DL3 genes (79% vs. 95%, p=.0054; 67% vs. 89%, p=.0032). Analysis of the KIR genotype in correlation with the corresponding KIR-L profile, deduced from HLA typing, revealed an increased frequency of unopposed 2DS2 (2DS2/C1 type, 37% vs.10%, p=.012) and 2DL2 (2DL2/C1 mismatch 37% vs 11%, p=.031) but these mismatches have a potentially opposing functional effect. Using sequence-specific PCR amplification and/or direct sequencing, we have genotyped DNA samples derived from our cohort who presented with different subtypes of PNH and studied single nucleotide polymorphisms (SNPs) in cytokine genes such as TNF-a (−308 G/A), TGF-b 1 (C/T codon 10, C/G codon 25), IL-10 (−1082 G/A), and IFN-g (+874 A/T) and immunomodulatory receptor genes like CTLA-4 exon 6 (+49 G/A), FcγRIIIa (158 F/V) and CD45-exons 6 (+138 A/G), and 4 (+54 A/G, +77 C/G). These SNPs are responsible for intrinsic differences in cytokine production and receptor function and can thereby influence immune physiologic and pathologic responses. PNH patients showed a significantly higher frequency of A/A polymorphisms at intron −1082 of the IL-10 gene consistent with a “low secretor phenotype” (36% PNH (N=50) vs 13.8% controls (N=363), p=0.0001). In contrast to a few smaller studies, no association with any other SNP tested was found. To establish whether this finding translates into functional consequences, we examined basal and PMA-induced IL-10 secretion in PNH patients andcontrols. Upon induction, IL-10 production increased in controls (N=5, 14.11± 9.8), while in general, stimulation resulted in a much weaker IL-10 response in PNH patients (N=6, 4.51± 2.16). However, when compared genotypic IL-10 “low secretors” (−1082 A/A) among PNH patients showed significantly lower induction levels of IL-10 (N=2, 3.1± 1.58) as compared to PNH patients with “normal/high secretor” genotypes (−1082 A/G,G/G) ((N=4, 5.22 +/−3.1). The low secretor IL-10 genotype may correspond to the exaggerated TH1 response in PNH and in general supports the notion that complex inherited traits may exist that genetically determine propensity to PNH evolution.

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