Project description:We have engineered the thermostable KlenTaq DNA polymerase variant called RIV A8 that produces error signatures specific for 5-methylcytosine (5mC) and 5-hydroxycytosine (5hmC) without prior chemical treatment of the DNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish C from 5mC and C from 5hmC in DNA templates generated by PCR using modified dCTPs (unmodified by using dCTP, methylated by using d5mCTP, hydroxymethylated by using d5hmCTP).
Project description:Genome editing was conducted on a t(3;8) K562 model to investigate the effects of deleting different modules or CTCF binding sites within the MYC super-enhancer. To check mutations after targeting with CRISPR-Cas9 we performed amplicon sequencing using the Illumina PCR-based custom amplicon sequencing method using the TruSeq Custom Amplicon index kit (Illumina). The first PCR was performed using Q5 polymerase (NEB), the second nested PCR with KAPA HiFi HotStart Ready mix (Roche). Samples were sequenced paired-end (2x 250bp) on a MiSeq (Illumina).
Project description:We have engineered thermostable KlenTaq DNA polymerase variants called RIII H20, RIV A8 and RIV D15 that produce error signatures specific for 5-methylcytosine (5mC) without prior chemical treatment of the DNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish C from 5mC in DNA templates generated by PCR (unmodified and methylated using the CpG Methyltransferase (M.SssI)). Finally, RIV A8 was used to detect 5mC in HeLa human genomic DNA (native methylation and supplementary methylated using the CpG Methyltransferase (M.SssI))
2024-09-09 | GSE233599 | GEO
Project description:Amplicon-based NGS to detect Cutibacterium acnes phylotypes
| PRJNA801471 | ENA
Project description:Amplicon NGS to determine staphylococcal species on human skin
Project description:We have engineered an RT-active DNA polymerase variant called RT-KTq I614Y that produces error RT‑signatures specific for pseudouridine (Ψ) without prior chemical treatment of the RNA samples. These signatures are amplified during DNA amplicon library preparation and are detected by NGS. This method was applied to distinguish U from Ψ in RNA oligonucleotides (modified or unmodified) used in the previous polymerase screening and oligonucleotides which are designed from the human 18S rRNA at the position around 1445. Finally, RT-KTq I614Y was used to detect Ψ55 in tRNAGly(GCC) from Saccharomyces cerevisiae wildtype compared to data from tRNAGly(GCC) from S. cerevisiae pus4Δ.
Project description:Milk fat globule EGF factor 8 (MFGE8) is a glycoprotein which plays a crucial role in mammary gland remodeling. Our group previously identified MFGE8 as a marker associated with high milk yielding cows. Here, we generated MFGE8 knock-out buffalo mammary epithelial cells (BuMEC) via CRISPR-cas9 technology to decipher its role in lactation. gRNA3 reduced MFGE8 expression with good efficiency which was confirmed at transcriptomic and proteomic level and the stable knock-out cells obtained were named mfge8-/-/gRNA3. The amplicon sequencing of the edited region using next generation sequencing (NGS) showed that 54% of total reads showed indels, 3-4 bp upstream to PAM site in 2nd exon. A total 4282 proteins were identified when proteome level changes were examined and 178 were found to be differentially expressed above and below a threshold of ≥1.5 and ≤0.6. Major DEPs were found to be associated with regulation of hydrolase activity, endopeptidase activity and cytoskeletal organization and some DEPs including FABP3, FABP4, FABP5, KNG1, MT2A, CD82 and SERPINH1 belonged to genes associated with milk synthesis. To the best of our knowledge, this is the first study which provides a comprehensive proteome profile of MFGE8 knockout BuMEC and explores downstream effects of disruption of MFGE8 gene.