Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a newly emerging virus well known as the major cause of the worldwide pandemic due to Coronavirus Disease 2019 (COVID-19). Major breakthroughs in the Next Generation Sequencing (NGS) field were elucidated following the first release of a full-length SARS-CoV-2 genome on the 10 January 2020, with the hope of turning the table against the worsening pandemic situation. Previous studies in respiratory virus characterization require mapping of raw sequences to the human genome in the downstream bioinformatics pipeline as part of metagenomic principles. Illumina, as the major player in the NGS arena, took action by releasing guidelines for improved enrichment kits called the Respiratory Virus Oligo Panel (RVOP) based on a hybridization capture method capable of capturing targeted respiratory viruses, including SARS-CoV-2; therefore, allowing a direct map of raw sequences data to SARS-CoV-2 genome in downstream bioinformatics pipeline. Consequently, two bioinformatics pipelines emerged with no previous studies benchmarking the pipelines. This study focuses on gaining insight and understanding of target enrichment workflow by Illumina through the utilization of different bioinformatics pipelines named as ‘Fast Pipeline’ and ‘Normal Pipeline’ to SARS-CoV-2 strains isolated from Yogyakarta and Central Java, Indonesia. Overall, both pipelines work well in the characterization of SARS-CoV-2 samples, including in the identification of major studied nucleotide substitutions and amino acid mutations. A higher number of reads mapped to the SARS-CoV-2 genome in Fast Pipeline and merely were discovered as a contributing factor in a higher number of coverage depth and identified variations (SNPs, insertion, and deletion). Fast Pipeline ultimately works well in a situation where time is a critical factor. On the other hand, Normal Pipeline would require a longer time as it mapped reads to the human genome. Certain limitations were identified in terms of pipeline algorithm, whereas it is highly recommended in future studies to design a pipeline in an integrated framework, for instance, by using NextFlow, a workflow framework to combine all scripts into one fully integrated pipeline.

The coordinated activation of DNA replication origins is important for efficient DNA synthesis and genome stability. S-phase cyclin dependent kinases (CDKs) together with CDC7 kinase, are essential to origin activation by converting the pre-replicative complex into a fully active helicase. To identify genes that tune DNA replication, we have performed a chemo-genetic genome-wide CRISPR-KO screen with cells challenged with the CDC7 inhibitor XL413. By developing a methodology based on genetic coessentiality to functionally cluster the hits, we uncover the transcriptional CDK8/CCNC kinase in a cluster with replication initiation factors. We find that CDK8 depletion further reduces the rate of DNA synthesis imposed by CDC7 inhibitors. DNA fibre experiments provide compelling evidence that CDK8 and CDC7 cooperate in origin activation. Suppression of DNA synthesis by CDK8 inhibition requires the binding of CDK8/CCNC to the MDM2 Binding Protein (MTBP) and we show that CDC7 and CDK8 individually contribute to the phosphorylation of MCM4 subunit of the replicative helicase. Thus, this work identifies CDK8 as the third protein kinase directly involved in origin activation in human cells.

Introduction- Colorectal cancer (CRC) is a development of abnormal cells either in colon or rectum. CRC considered being the 3rd leading cause of death in 2018 only behind lung and breast cancer. It first arises during pre-cancerous stages called as polyps. The detection and removal of polyp is important to increase the survival rate of patient. Various method of polyp detection are available. However, only colonoscopy remains the gold standard in detection and removal of polyps. Several studies showed how Artificial Intelligence (AI) used in colonoscopy area particularly in detecting polyps, assessing physicians and predicting patient with high risk of CRC. The aim of this study is to describe the involvement of AI in colonoscopy and its impact in reducing the Materials and methods– Search for journal articles conducted between May and June 2016 from various resources including PubMed and Google Scholar. 6 research journals were reviewed and all the advantages and limitations were discussed throughout this study. Results– Various study showed that AI able to improve medical diagnostic of CRC in several ways, including in the improvement of adenoma detection rate (ADR) in terms of medical diagnostic, finding physicians associated with high Adenoma Detection Rate (ADR) and predicting patients with high risk of CRC. In addition, the use of AI in colonoscopy also associated with limitations including require large amount of datasets and advance computational resources in order to generate accurate output. Conclusion– The utilization of AI in colonoscopy shows how it able to improve the diagnosis accuracy and survival rate of patients associated with CRC despite several limitations that were identified during the study. However in the future, instead of allowing it to fully automatically conducting diagnosis, it still needs to be accompanied by physicians conducting the operation as there is no hundred percent perfect algorithms.

The coordinated activation of DNA replication origins is important for efficient DNA synthesis and genome stability. S-phase cyclin-dependent kinases (CDKs), together with CDC7 kinase, are essential for origin activation by converting the pre-replicative complex into a fully active helicase. Here, in order to identify genes that tune DNA replication, we performed a chemogenetic genome-wide CRISPR-KO screen in cells challenged with the CDC7 inhibitor XL413. By developing a methodology based on genetic co-essentiality to functionally cluster the hits, we uncover the transcriptional CDK8/CCNC kinase in a cluster with replication initiation factors. Depletion of CDK8 further reduces the rate of DNA synthesis imposed by CDC7 inhibitors. DNA fibre experiments further support that CDK8 and CDC7 cooperate in origin activation. Suppression of DNA synthesis by CDK8 inhibition requires the binding of CDK8/CCNC to the MDM2 Binding Protein (MTBP), and we show that CDC7 and CDK8 associate with the MCM complex and individually contribute to the phosphorylation of the replicative helicase. In conclusion, this work adds CDK8 to the list of kinases directly involved in origin activation in human cells.

Pooled genome-wide CRISPR-Cas9 knockout (CRISPR-KO) screening is a powerful approach for discovering new biology and identifying genetic vulnerabilities in cancers. This approach uses the Cas9 nuclease in combination with sgRNA libraries, typically consisting of 4-8 sgRNAs to induce mutations in each target gene. A critical assumption is that the effect of each sgRNA is solely due to Cas9 editing of the target gene. However, libraries can contain sgRNAs that direct Cas9 to multiple locations, thus potentially introducing bias into gene hit lists and leading to flawed biological hypotheses. Here we have developed GuideRefine, a pipeline to detect multi-targeting and off-targeting sgRNAs. GuideRefine outputs a virtual refined sub-library containing only on-target sgRNAs. Using GuideRefine with T2T-CHM13 as the reference genome, we surveyed the Brunello, TKOv3, Yusa, Avana, and Jacquere libraries, finding that ~7.5% to ~16% of sgRNAs are potentially problematic. We confirmed that multi-targeting sgRNAs disproportionately impair cell fitness and that sgRNAs aligning to more than one location with a single mismatch can also reduce fitness, although to a lesser extent. After flagging problematic sgRNAs and creating virtual ″on-target only″ sub-libraries, ~10% to ~16% of genes lose critical representation (< 3 sgRNAs per gene). Intriguingly, a set of 467 genes, characterised by short CDS length and lower PAM site density, have fewer than three sgRNAs in all sub-libraries, suggesting they cannot be well-targeted using current libraries. We anticipate that GuideRefine, together with caution in assessing the effects of problematic sgRNAs, will help prioritise biologically relevant hits.