Preparing Gene of interest for GateWay cloning (2 step PCR process) v2

GateWay recombination cloning is achieved by flanking your gene of interest with GateWay attachment sites. In our case attB1 and attB2. Those sites are added to the PCR product via primers with 5' extensions. Since those primes create 31 bp and 30 bp 5' primer extensions respectively, plus about 20 bp of actual binding primer sequence it becomes expensive fast if you need 2 x ~50 bp primers for every GOI. We therefore use a 2 step PCR process to attach GateWay attB1 and attB2 sites. We first run a gene specific PCR with primers carrying short 5' extesions, and then a second PCR utilizing universal GateWay primers which bind to the short extension of the first PCR product to create the full attB1 and attB2 sites. This protocol has been adapted from: 2-STEP GATEWAY PCR EXPERIMENTS

A Simplified Gibson Assembly Method for Site Directed Mutagenesis Using Non-Gibson Primers

Background: Site-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired. Method: We present a versatile and simple method to efficiently introduce a variety of mutation schemes using the Gibson-assembly without the need for unique Gibson primers. The method entails use of standard SDM primers (shorter and completely overlapping in sequences in contrast to Gibson primers) that are separately employed with common primer (~25 bps long) for amplification of fragments flanking the site of mutagenesis, followed by rapid amplification of the Gibson-assembled product for added visualization and sequencing steps for ensuring high success rates.Results: We find that assembly of the fragments via the Gibson reaction mixture is attainable within as short as 15 minutes, despite the need for extensive digestion of the DNA (by exonuclease) past the entire SDM primer sequence (to expose non-clashing overlap between the fragments). We also find that the amount of the assembled Gibson product is too low to be visualized and assessed on standard agarose gel. We thereby introduce a short amplification step (by use of the same short primers initially employed) to 1) easily resolve whether the product (only the correct size can yield a product) has been obtained, and 2) for isolation of product for DNA-sequencing (to assess whether mutation(s) have been introduced). No other SDM method enables assessment of mutagenesis prior completion of the process. Conclusion: We employ our approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically in DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). The entire protocol spans only four days, requires minimal primers sets (as well as can be used with most in-house primers) and provides very high yields and success rates (>98%).

Running the Titan_Illumina_PE Workflow on Terra.bio v1

The Titan_Illumina_PE workflow is a part of the Public Health Viral Genomics Titan series for SARS-CoV-2 genomic characterization. Titan_Illumina_PE was written specifically to process Illumina paired-end (PE) read data. Input reads are assumed to be the product of sequencing tiled PCR-amplicons designed for the SARS-CoV-2 genome. The most common read data analyzed by the Titan_Illumina_PE workflow are generated with the ARTIC V3 protocol. However, alternative primer schemes such as the QIAseq Primer Panel are also suitable for this workflow. The primer sequence coordinates of the PCR scheme utilized must be provided in BED format along with the raw Illumina read data. Upon initiating a Titan_Illumina_PE job, the input primer scheme coordinates and raw paired-end Illumina read data provided for each sample will be processed to perform consensus genome assembly, infer the quality of both raw read data and the generated consensus genome, and assign lineage or clade designations as outlined in the Titan_Illumina_PE data workflow diagram below. Additional technical documentation for the Titan_Illumina_PE workflow is available at: https://public-health-viral-genomics-theiagen.readthedocs.io/en/latest/titan_workflows.html#titan-workflows-for-genomic-characterization Required input data for Titan Illumina PE: Illumina paired-end read data (forward and reverse FASTQ files per sample) Primer sequence coordinates of the PCR scheme utilized in BED file format Video Instruction: Theiagen Genomics: Titan Genomic Characterization https://www.youtube.com/watch?v=zP9I1r6TNrw Theiagen Genomics: Titan Outputs QC https://www.youtube.com/watch?v=Amb-8M71umw For technical assistance please contact us at: [email protected]

Running the Titan_ONT Workflow on Terra.bio v1

The Titan_ONT workflow is a part of the Public Health Viral Genomics Titan series for SARS-CoV-2 genomic characterization. Titan_ONT was written specifically to process basecalled and demultiplexed Oxford Nanopore Technology (ONT) read data. Input reads are assumed to be the product of sequencing ARTIC V3 tiled PCR-amplicons designed for the SARS-CoV-2 genome. Upon initiating a Titan_ONT run, input read data provided for each sample will be processed to perform consensus genome assembly, infer the quality of both raw read data and the generated consensus genome, and assign lineage or clade designations as outlined in the Titan_ONT data workflow diagram below. Additional technical documentation for the Titan_ONT workflow is available at: https://public-health-viral-genomics-theiagen.readthedocs.io/en/latest/titan_workflows.html#titan-workflows-for-genomic-characterization Required input data for Titan_ONT: Basecalled and demultiplexed ONT read data files (single FASTQ file per sample) Primer sequence coordinates of the PCR scheme utilized in BED file format Titan_ONT has not been written to process FAST5 files Video Instruction: Theiagen Genomics: Titan Genomic Characterization https://www.youtube.com/watch?v=zP9I1r6TNrw Theiagen Genomics: Titan Outputs QC https://www.youtube.com/watch?v=Amb-8M71umw For technical assistance please contact us at: [email protected]

Running the Titan_ClearLabs Workflow on Terra.bio v1

The Titan_ClearLabs workflow is a part of the Public Health Viral Genomics Titan series for SARS-CoV-2 genomic characterization. Titan_CleanLabs was written to process Clear Labs read data for SARS-CoV-2 ARTIC V3 amplicon sequencing. Upon initiating a Titan_ClearLabs run, input read data provided for each sample will be processed to perform consensus genome assembly, infer the quality of both raw read data and the generated consensus genome, and assign lineage or clade designations as outlined in the Titan_ClearLabs data workflow below. Additional technical documentation for the Titan_ClearLabs workflow is available at: https://public-health-viral-genomics-theiagen.readthedocs.io/en/latest/titan_workflows.html#titan-clearlabs Required input data for Titan_ClearLabs: Cear Labs FASTQ read files (single FASTQ file per sample) Primer sequence coordinates of the PCR scheme utilized in BED file format Video Instruction: Theiagen Genomics: Titan Genomic Characterization https://www.youtube.com/watch?v=zP9I1r6TNrw Theiagen Genomics: Titan Outputs QC https://www.youtube.com/watch?v=Amb-8M71umw For technical assistance, please contact us at: [email protected]

Study of myocillin gene variants alleles in primary open angle glaucoma patients and their first degree relatives in North West Rajasthan, India

Glaucoma is defined as progressive optic neuropathy leading to irreversible blindness if not treated on time. Primary open angle glaucoma (POAG) is most common form of glaucoma. Mutations in myocilin gene (MYOC) account for 2–4% of POAG cases. To identify and evaluate MYOC variants alleles among patients with POAG and their healthy first degree relatives. 66 POAG patients and 26 healthy first degree relatives recruited for study. All patients underwent complete ophthalmic examination followed by genomic DNA (deoxyribonucleic acid) isolation from peripheral blood and quantification of DNA on spectrophotometer. All samples were amplified with each primer by PCR (Polymerase Chain Reaction) technique and amplified DNA and primer sequence checked again by electrophoresis for confirmation of specified MYOC gene mutation. We identified a known MYOC missense mutation, Pro370leu in 16 POAG cases and found consistent genotypic but not phenotypic correlation in 4 of their first degree relatives. Out of 16 cases, pathogenic MYOC gene variant was found in 12 adult onset POAG, 3 juvenile onset POAG, and 1 case of OHT. This study is first of its kind in North India. Our study showed frequency of MYOC gene mutation in POAG cases was 24.24% which is much higher than found elsewhere in India and other countries (2-5%). Frequency of transmission of pathogenic MYOC gene variant in first degree relatives was 25%. The future outcome of our study is promising since early diagnosis and management of high risk family members is possible.

Impact of gamma irradiation pretreatment on biochemical and molecular responses of potato growing under salt stress

Background Previous literatures revealed that gamma rays have an increasing effect on salt tolerance in different plants. In vitro experiment was conducted to study the effect of gamma rays (20 Gray) on salt tolerance of four potato cultivars (Lady Rosetta, Diamante, Gold, and Santana). Results Gamma-treated Santana plantlets were more tolerant to salinity as compared to other cultivars. It showed a significant increment of fresh weight (250% over the untreated). Gamma-treated plantlets of Lady Rosetta, Diamante, and Gold showed higher activity of peroxidase (POD) and polyphenol oxidase (PPO). Isoenzymes analysis showed an absence of POD 3, 4, and 5 in Gold plantlets. The dye of most PODs and PPOs bands were denser (more active) in gamma-treated plantlets of Santana as compared to other cultivars. Both gamma-treated and untreated plantlets showed the absence of PPO1 in Lady Rosetta and Diamante, and PPO 3, 4, and 5 in Gold plantlets. Genetic marker analysis using ISSR with six different primers showed obvious unique negative and positive bands with different base pairs in mutant plantlets as compared to the control, according to primer sequence and potato genotype. The 14A primer was an efficient genetic marker between mutated and unmutated potato genotypes. Santana had a unique fingerprint in the 1430-pb site, which can be a selectable marker for the cultivar. An increment in genetic distance between Gold cultivar and others proved that the mutation was induced because of gamma rays. Conclusion We assume that irradiation of potato callus by 20-Gy gamma rays is an effective process for inducing salt resistance. However, this finding should be verified under field conditions. Graphic Abstract

Identification of the Novel HLA-A*11:335 Allele, a Rare Interlocus Recombination Involving HLA-A*11:01:01:01 and HLA-H*02:07/14/18 Alleles With Nanopore Sequencing, in a Volunteer From the China Marrow Donor Program

Background: The major histocompatibility complex in humans includes three classical class I loci (A, B and C), which are important biomarkers for transplant of organs and hematopoietic stem cells. In the MHC, polymorphism is known to be extremely high while interlocus recombination is rare. We report a rare interlocus recombination between HLA-A and HLA-H, which was analyzed using next generation sequencing and nanopore sequencing. Results: In the sample, the genotypes of HLA-A, B, C, DRB1 and DQB1 were firstly phased with methods of sequence-specific primer, sequence-specific oligonucleotide, Sanger’s sequencing and NGS; however, HLA-A could not be phased. Nanopore sequencing was finally utilized to distinguish the sequence of the novel allele. Finally, the novel HLA-A*11:335 allele was identified as an interlocus recombination involving HLA-A*11:01:01:01 and HLA-H*02:07/14/18 alleles; this was mainly achieved by nanopore sequencing. Conclusions: The identification of the interlocus recombination indicated that nanopore sequencing may be the most precise method for HLA typing. Interlocus recombination has been identified as one of the mechanisms involved in the generation of novel HLA alleles.

Genetic polymorphism of myostatin (MSTN) in Nigerian sheep breeds

Myostatin (MSTN) also known as growth differentiation factor 8 (GDF-8) has been implicated to play an important role in growth regulation, and it is a candidate gene in marker assisted selection (MAS). This study was carried out to identify the polymorphism of MSTN gene as a genetic marker for growth traits in Nigerian indigenous sheep. Genomic DNA (gDNA) was extracted from blood samples of Balami, Yankasa, Uda and West African Dwarf (WAD) breeds of sheep. Parts of 5’UTR, intron and exon1 (614bp) was amplified using a primer sequence designed by FastPCR-primer software. The amplicons were digested with restriction enzyme HaeIII and the fragments produced were stained with luminescent dye and run on gel electrophoresis. The genetic structure of the sampled population was investigated after analysis with POPGENE32 software. The HaeIII digested results showed that Myostatin has three polymorphs (AA, AB and BB), controlled by two alleles (A and B), with B having a higher allelic frequency (82.84%) and BB genotype has the highest frequency of 73%. The sampled population showed a deviation from Hardy-Weinberg equilibrium (p<0.05) while the F-statistics results of the Nigerian breeds of sheep showed the breeds are genetically identical (33.40%) within them. The genetic distance matrix established that Uda and Yankasa show the greatest distant (3.00%) while Uda and WAD are almost identical (99.85%). The four breeds of sheep studied showed polymorphism for Myostatin gene in the intron 1 and exon 1. Myostatin, therefore, could be considered a candidate gene for MAS

Prediction of PCR amplification from primer and template sequences using recurrent neural network

We have developed a novel method to predict the success of PCR amplification for a specific primer set and DNA template based on the relationship between the primer sequence and the template. To perform the prediction using a recurrent neural network, the usual double-stranded formation between the primer and template nucleotide sequences was herein expressed as a five-lettered word. The set of words (pseudo-sentences) was placed to indicate the success or failure of PCR targeted to learn recurrent neural network (RNN). After learning pseudo-sentences, RNN predicted PCR results from pseudo-sentences which were created by primer and template sequences with 70% accuracy. These results suggest that PCR results could be predicted using learned RNN and the trained RNN could be used as a replacement for preliminary PCR experimentation. This is the first report which utilized the application of neural network for primer design and prediction of PCR results.