Benchmarking and optimization of a high‐throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development

2021 ◽  
pp. 2000548
Author(s):  
Joost Groot ◽  
Yizhou Zhou ◽  
Eric Marshall ◽  
Patrick Cullen ◽  
Thomas Carlile ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Variant ◽  
Cell Line Development ◽  
Variant Analysis

scholarly journals CDK10 Regulates Gastric Cancer Metastasis By Inhibiting lncRNA-C5ORF42-5 Via Phosphorylation Level of AKT/ERK

2021 ◽  
Author(s):  
Zi-Jian Deng ◽  
Dong-Wen Chen ◽  
Xi-Jie Chen ◽  
Jia-Ming Fang ◽  
Liang Xv ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Cells ◽  
Cell Line ◽  
High Throughput ◽  
Cancer Metastasis ◽  
High Throughput Sequencing ◽  
Gastric Cancer Cells ◽  
Related Proteins

Abstract Background: Gastric cancer is the fourth most common malignant disease. Both CDK10 and long noncoding RNAs (lncRNAs) have been found to exert biological functions in multiple cancers. However, it is still unclear whether CDK10 represses tumor progression in gastric cancer by reducing potential targeting lncRNAs.Methods: The functions of CDK10 and lncRNA-C5ORF42-5 in proliferation, invasion and migration were assessed by MTS assays, colony formation assays, cell cycle and apoptosis assays, Transwell assays, wound healing assays and animal experiments. We used high-throughput sequencing to confirm the existence of lncRNA-C5ORF42-5 and quantitative real-time PCR was used to evaluate lncRNA expression. Then, with RNA-seq sequencing as well as GO function and KEGG enrichment analysis, we identified the signaling pathways in which lncRNA-C5ORF42-5 was involved in gastric cancer. Finally, western blotting was used to identify the genes regulated by lncRNA-C5ORF42-5.Results: Our results showed that CDK10 is expressed at relatively low levels in gastric cancer cell lines and inhibits the progression of gastric cancer cells both in vitro and in vivo. Next, based on high-throughput sequencing, we identified a novel lncRNA, lncRNA-C5ORF42-5, in the stable CDK10-overexpressing cell line compared with the CDK-knockdown cell line and their controls. Additionally, we confirmed that lncRNA-C5ORF42-5 acts as an oncogene to promote metastasis in gastric cancer in vitro and in vivo. We then ascertained that lncRNA-C5ORF42-5 is a major contributor to the function of CDK10 in gastric cancer metastasis by upregulating lncRNA-C5ORF42-5 to reverse the effects of CDK10 overexpression. Finally, we explored the mechanism by which lncRNA-C5ORF42-5 overexpression affects gastric cancer cells to elucidate whether lncRNA-C5ORF42-5 may increase the activity of the SMAD pathway of BMP signaling and promote the expression of EMT-related proteins, such as E-cadherin. Additionally, overexpression of lncRNA-C5ORF42-5 affected the phosphorylation levels of AKT and ERK.Conclusion: Our findings suggest that CDK10 overexpression represses gastric cancer tumor progression by reducing lncRNA-C5ORF42-5 and hindering activation of the related proteins in metastatic signaling pathways, which provides new insight into developing effective therapeutic strategies in the treatment of metastatic gastric cancer.

scholarly journals High-throughput Sequencing for Species Authentication and Contamination Detection of 63 Cell Lines

2021 ◽  
Author(s):  
Oliver Lung ◽  
Rebecca Candlish ◽  
Michelle Nebroski ◽  
Peter Kruckiewicz ◽  
Cody Buchanan ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Classical Swine Fever ◽  
Virus Genome ◽  
Cox1 Gene ◽  
Species Identity ◽  
Cell Line Authentication ◽  
Dna And Rna

Abstract Cell lines are widely used in research and for diagnostic tests and are often shared between laboratories. Lack of cell line authentication can result in the use of contaminated or misidentified cell lines, potentially affecting the results from research and diagnostic activities. Cell line authentication and contamination detection based on metagenomic high-throughput sequencing (HTS) was tested on DNA and RNA from 63 cell lines available at the Canadian Food Inspection Agency’s National Centre for Foreign Animal Disease. Through sequence comparison of the cytochrome c oxidase subunit 1 (COX1) gene, the species identity of 53 cell lines was confirmed, and eight cell lines were found to show a greater pairwise nucleotide identity in the COX1 sequence of a different species within the same expected genus. Two cell lines, LFBK-αvβ6 and SCP-HS, were determined to be composed of cells from a different species and genus. Mycoplasma contamination was not detected in any cell lines. However, several expected and unexpected viral sequences were detected, including part of the classical swine fever virus genome in the IB-RS-2 Clone D10 cell line. Metagenomics-based HTS is a useful laboratory QA tool for cell line authentication and contamination detection that should be conducted regularly.

scholarly journals IROme, a New High-Throughput Molecular Tool for the Diagnosis of Inherited Retinal Dystrophies

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Daniel F. Schorderet ◽  
Alexandra Iouranova ◽  
Tatiana Favez ◽  
Leila Tiab ◽  
Pascal Escher
Keyword(s):  
Retinitis Pigmentosa ◽  
Molecular Diagnosis ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Sequence Variant ◽  
Sequence Variants ◽  
Sequencing Platform ◽  
Capture Assay ◽  
Retinal Dystrophies ◽  
Exon Capture

The molecular diagnosis of retinal dystrophies is difficult because of the very important number of genes implicated and is rarely helped by genotype-phenotype correlations. This prompted us to develop IROme, a custom designed in solution-based targeted exon capture assay (SeqCap EZ Choice library, Roche NimbleGen) for 60 retinitis pigmentosa-linked genes and three candidate genes (942 exons). Pyrosequencing was performed on a Roche 454 GS Junior benchtop high-throughput sequencing platform. In total, 23 patients affected by retinitis pigmentosa were analyzed. Per patient, 39.6 Mb were generated, and 1111 sequence variants were detected on average, at a median coverage of 17-fold. After data filtering and sequence variant prioritization, disease-causing mutations were identified inABCA4,CNGB1,GUCY2D,PROM1,PRPF8,PRPF31,PRPH2,RHO,RP2, andTULP1for twelve patients (55%), ten mutations having never been reported previously. Potential mutations were identified in 5 additional patients, and in only 6 patients no molecular diagnosis could be established (26%). In conclusion, targeted exon capture and next-generation sequencing are a valuable and efficient approach to identify disease-causing sequence variants in retinal dystrophies.

High-throughput sialylation measurement using lectins on an Octet platform for clone screening

2016 ◽  
Vol 8 (39) ◽  
pp. 7193-7198 ◽  
Author(s):  
Kanvasri N. Jonnalagadda ◽  
Lam Raga A. Markely ◽  
Bing Guan ◽  
Christina Alves ◽  
Shashi Prajapati
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
Sialic Acids ◽  
Cell Line Development ◽  
Support Cell ◽  
High Throughput Method

A high-throughput method to quantify sialic acids was developed on a ForteBio Octet QK384 platform to support cell-line development.

scholarly journals MIP-MAP: High Throughput Mapping of Caenorhabditis elegans Temperature Sensitive Mutants via Molecular Inversion Probes

2017 ◽  
Author(s):  
CA Mok ◽  
V Au ◽  
OA Thompson ◽  
ML Edgley ◽  
L Gevirtzman ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Single Molecule ◽  
High Throughput Sequencing ◽  
Essential Genes ◽  
Model Organisms ◽  
Temperature Sensitive ◽  
Sequence Variant ◽  
Mutant Selection ◽  
Molecular Inversion Probes

AbstractTemperature sensitive (TS) alleles are important tools for the genetic and functional analysis of essential genes in many model organisms. While isolating TS alleles is not difficult, determining the TS-conferring mutation can be problematic. Even with whole-genome sequencing (WGS) data there is a paucity of predictive methods for identifying TS alleles from DNA sequence alone. We assembled 173 TS lethal mutants of Caenorhabditis elegans and used WGS to identify several hundred mutations per strain. We leveraged single molecule molecular inversion probes (MIPs) to sequence variant sites at high depth in the cross-progeny of TS mutants and a mapping strain with identified sequence variants but no apparent phenotypic differences from the reference N2 strain. By sampling for variants at ~1Mb intervals across the genome we genetically mapped mutant alleles at a resolution comparable to current standards in a process we call MIP-MAP. The MIP-MAP protocol, however, permits high-throughput sequencing of multiple TS mutation mapping libraries at less than 200K reads per library. Using MIP-MAP on a subset of TS mutants, via a competitive selection assay and standard recombinant mutant selection, we defined TS-associated intervals of 3Mb or less. Our results suggest this collection of strains contains a diverse library of TS alleles for genes involved in development and reproduction. MIP-MAP is a robust method to genetically map mutations in both viable and essential genes. The MIPs protocol should allow high-throughput tracking of genetic variants in any mixed population.

scholarly journals High-Throughput MicroRNA Profiles of Permissive Madin-Darby Canine Kidney Cell Line Infected with Influenza B Viruses

Viruses ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 986
Author(s):  
Saengchoowong ◽  
Khongnomnan ◽  
Poomipak ◽  
Praianantathavorn ◽  
Poovorawan ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Mdck Cells ◽  
Computational Prediction ◽  
Influenza B ◽  
Madin Darby Canine Kidney ◽  
High Throughput Analysis ◽  
Darby Canine Kidney ◽  
Canine Kidney

Victoria and Yamagata lineages of influenza B viruses are globally circulating in seasonal epidemics. Madin–Darby canine kidney (MDCK) cells are permissive for viral isolation and vaccine manufacture. Nevertheless, the interplay between influenza B viruses and host microRNAs has not been investigated in this cell line. Therefore, the present study aims at high-throughput analysis of canine microRNA profile upon infection of influenza B viruses. Briefly, MDCK cells were infected with Victoria or Yamagata lineage at MOI of 0.01. After being harvested at 6, 12 and 24 h post infection, microRNAs were subjected to high-throughput sequencing based on MiSeq platform (Illumina). The results demonstrated that five microRNAs including cfa-miR-197, cfa-miR-215, cfa-miR361, cfa-miR-1841, and cfa-miR-1842 were overexpressed in both Victoria and Yamagata lineage infections. Interestingly, computational prediction showed that karyopherin alpha 6 (KPNA6) was targeted by cfa-miR-197 and cfa-miR-215. Moreover, the binding sites of both microRNAs were assessed by 3′-UTR reporter assay. The results showed that only cfa-miR-197 could bind to the target sites of KPNA6, leading to suppressing luciferase activity. Additionally, silencing of KPNA6 was confirmed by overexpression of cfa-miR-197. This study provides canine microRNA responses to seasonal influenza B viruses, suggesting that virus-mediated microRNAs might play crucial roles in host gene regulation.

scholarly journals Differential binding cell-SELEX method to identify cell-specific aptamers using high-throughput sequencing

2018 ◽  
Author(s):  
Karlis Pleiko ◽  
Liga Saulite ◽  
Vadims Parfejevs ◽  
Karlis Miculis ◽  
Egils Vjaters ◽  
...  
Keyword(s):  
Cell Line ◽  
High Throughput ◽  
Negative Selection ◽  
Sanger Sequencing ◽  
High Throughput Sequencing ◽  
Kidney Tissue ◽  
Live Cells ◽  
Aptamer Selection ◽  
Differential Binding ◽  
Binding Cell

ABSTRACTAptamers have in recent years emerged as a viable alternative to antibodies. High-throughput sequencing (HTS) has revolutionized aptamer research by increasing the number of reads from a few (using Sanger sequencing) to millions (using an HTS approach). Despite the availability and advantages of HTS compared to Sanger sequencing, there are only 50 aptamer HTS sequencing samples available on public databases. HTS data in aptamer research are primarily used to compare sequence enrichment between subsequent selection cycles. This approach does not take full advantage of HTS because the enrichment of sequences during selection can be due to inefficient negative selection when using live cells. Here, we present a differential binding cell-SELEX (systematic evolution of ligands by exponential enrichment) workflow that adapts theFASTAptamertoolbox and bioinformatics tooledgeR, which are primarily used for functional genomics, to achieve more informative metrics about the selection process. We propose a fast and practical high-throughput aptamer identification method to be used with the cell-SELEX technique to increase the aptamer selection rate against live cells. The feasibility of our approach is demonstrated by performing aptamer selection against a clear cell renal cell carcinoma (ccRCC) RCC-MF cell line using the RC-124 cell line from healthy kidney tissue for negative selection.

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