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Single cell RNAseq has been a big leap in many areas of biology. Rather than investigating gene expression on a whole organism level, this technology enables scientists to get a detailed look at rare single cells or within their cell population of interest. The field is growing, and many new methods appear each year. We compared methods utilized in our core facility: Smart-seq3, PlexWell, FLASH-seq, VASA-seq, SORT-seq, 10X, Evercode, and HIVE. We characterized the equipment requirements for each method. We evaluated the performances of these methods based on detected features, transcriptome diversity, mitochondrial RNA abundance and multiplets, among others and benchmarked them against bulk RNA sequencing. Here, we show that bulk transcriptome detects more unique transcripts than any single cell method. While most methods are comparable in many regards, FLASH-seq and VASA-seq yielded the best metrics, e.g., in number of features. If no equipment for automation is available or many cells are desired, then HIVE or 10X yield good results. In general, more recently developed methods perform better. This also leads to the conclusion that older methods should be phased out, and that the development of single cell RNAseq methods is still progressing considerably. [ABSTRACT FROM AUTHOR]

With the advent of next-generation sequencing (NGS) methodologies, the total repertoires of B and T cells can be disclosed in much more detail than ever before. Even though many of these strategies do provide in-depth and high-resolution information of the immunoglobulin (IG) and/or T-cell receptor (TR) repertoire, one clear disadvantage is that the IG/TR profiles cannot be connected to individual cells. Single-cell technologies do allow to study the IG/TR repertoire at the individual cell level. This is especially relevant in cell samples in which much heterogeneity of the cell population is expected. By combining the IG/TR repertoire with transcriptome data, the reactivity of the B or T cell can be associated with activation or maturation stages. An additional advantage of such single-cell technologies is that the combination of both IG and both TR chains can be studied on a per cell basis, which better reflects the antigen receptor reactivity of cells. Here we present the ICELL8 single-cell method for the parallel analysis of the TR repertoire and transcriptome, which is especially useful in samples that contain relatively few cells., (© 2022. The Author(s).)

Background: Biomarkers predicting treatment response may be used to stratify pancreatic ductal adenocarcinoma (PDAC) patients for therapy. The aim of this study was to identify circulating tumor DNA (ctDNA) mutations that associate with tumor progression during FOLFIRINOX chemotherapy, and overall survival (OS)., Methods: Circulating cell-free DNA was analyzed with a 57 gene next-generation sequencing panel using plasma samples of 48 PDAC patients of all disease stages. Patients received FOLFIRINOX as initial treatment. Chemotherapy response was determined on CT scans as disease control ( n  = 30) or progressive disease ( n  = 18) within eight cycles of FOLFIRINOX, based on RECIST 1.1 criteria., Results: Detection of a TP53 ctDNA mutation before start of FOLFIRINOX [odds ratio (OR) 10.51, 95% confidence interval (CI) 1.40-79.14] and the presence of a homozygous TP53 Pro72Arg germline variant (OR 6.98, 95% CI 1.31-37.30) were predictors of early tumor progression during FOLFIRINOX in multivariable analysis. Five patients presented with the combination of a TP53 ctDNA mutation before start of FOLFIRINOX and the homozygous Pro72Arg variant. All five patients showed progression during FOLFIRINOX. The combination of the TP53 mutation and TP53 germline variant was associated with shorter survival (median OS 4.4 months, 95% CI 2.6-6.2 months) compared with patients without any TP53 alterations (median OS 13.0 months, 95% CI 8.6-17.4 months)., Conclusion: The combination of a TP53 ctDNA mutation before start of FOLFIRINOX and a homozygous TP53 Pro72Arg variant is a promising biomarker, associated with early tumor progression during FOLFIRINOX and poor OS. The results of this exploratory study need to be validated in an independent cohort., Competing Interests: Conflict of interest statement: The authors declare that there is no conflict of interest., (© The Author(s), 2021.)