Dataset Information

Assessing Transcriptome Quality in Patch-Seq Datasets.

ABSTRACT: Patch-seq, combining patch-clamp electrophysiology with single-cell RNA-sequencing (scRNAseq), enables unprecedented access to a neuron's transcriptomic, electrophysiological, and morphological features. Here, we present a re-analysis of five patch-seq datasets, representing cells from ex vivo mouse brain slices and in vitro human stem-cell derived neurons. Our objective was to develop simple criteria to assess the quality of patch-seq derived single-cell transcriptomes. We evaluated patch-seq transcriptomes for the expression of marker genes of multiple cell types, benchmarking these against analogous profiles from cellular-dissociation based scRNAseq. We found an increased likelihood of off-target cell-type mRNA contamination in patch-seq cells from acute brain slices, likely due to the passage of the patch-pipette through the processes of adjacent cells. We also observed that patch-seq samples varied considerably in the amount of mRNA that could be extracted from each cell, strongly biasing the numbers of detectable genes. We developed a marker gene-based approach for scoring single-cell transcriptome quality post-hoc. Incorporating our quality metrics into downstream analyses improved the correspondence between gene expression and electrophysiological features. Our analysis suggests that technical confounds likely limit the interpretability of patch-seq based single-cell transcriptomes. However, we provide concrete recommendations for quality control steps that can be performed prior to costly RNA-sequencing to optimize the yield of high-quality samples.

SUBMITTER: Tripathy SJ

PROVIDER: S-EPMC6187980 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Assessing Transcriptome Quality in Patch-Seq Datasets.

Tripathy Shreejoy J SJ Toker Lilah L Bomkamp Claire C Mancarci B Ogan BO Belmadani Manuel M Pavlidis Paul P

Frontiers in molecular neuroscience 20181008

Patch-seq, combining patch-clamp electrophysiology with single-cell RNA-sequencing (scRNAseq), enables unprecedented access to a neuron's transcriptomic, electrophysiological, and morphological features. Here, we present a re-analysis of five patch-seq datasets, representing cells from <i>ex vivo</i> mouse brain slices and <i>in vitro</i> human stem-cell derived neurons. Our objective was to develop simple criteria to assess the quality of patch-seq derived single-cell transcriptomes. We evaluat ...[more]

PMID: 30349457

Similar Datasets

Project description:BackgroundSingle-cell RNA sequencing (scRNA-seq) has quickly become one of the most dominant techniques in modern transcriptome assessment. In particular, 10X Genomics' Chromium system, with its high throughput approach, turn key and thorough user guide made this cutting-edge technique accessible to many laboratories using diverse animal models. However, standard pre-processing, including the alignment and cell filtering pipelines might not be ideal for every organism or tissue. Here we applied an alternative strategy, based on the pseudoaligner kallisto, on twenty-two publicly available single cell sequencing datasets from a wide range of tissues of eight organisms and compared the results with the standard 10X Genomics' Cell Ranger pipeline.ResultsIn most of the tested samples, kallisto produced higher sequencing read alignment rates and total gene detection rates in comparison to Cell Ranger. Although datasets processed with Cell Ranger had higher cell counts, outside of human and mouse datasets, these additional cells were routinely of low quality, containing low gene detection rates. Thorough downstream analysis of one kallisto processed dataset, obtained from the zebrafish pineal gland, revealed clearer clustering, allowing the identification of an additional photoreceptor cell type that previously went undetected. The finding of the new cluster suggests that the photoreceptive pineal gland is essentially a bi-chromatic tissue containing both green and red cone-like photoreceptors and implies that the alignment and pre-processing pipeline can affect the discovery of biologically-relevant cell types.ConclusionWhile Cell Ranger favors higher cell numbers, using kallisto results in datasets with higher median gene detection per cell. We could demonstrate that cell type identification was not hampered by the lower cell count, but in fact improved as a result of the high gene detection rate and the more stringent filtering. Depending on the acquired dataset, it can be beneficial to favor high quality cells and accept a lower cell count, leading to an improved classification of cell types.

Project description:High-throughput RNA sequencing (RNA-seq) has become an instrumental assay for the analysis of multiple aspects of an organism's transcriptome. Further, the analysis of a biological specimen's associated microbiome can also be performed using RNA-seq data and this application is gaining interest in the scientific community. There are many existing bioinformatics tools designed for analysis and visualization of transcriptome data. Despite the availability of an array of next generation sequencing (NGS) analysis tools, the analysis of RNA-seq data sets poses a challenge for many biomedical researchers who are not familiar with command-line tools. Here we present RNA CoMPASS, a comprehensive RNA-seq analysis pipeline for the simultaneous analysis of transcriptomes and metatranscriptomes from diverse biological specimens. RNA CoMPASS leverages existing tools and parallel computing technology to facilitate the analysis of even very large datasets. RNA CoMPASS has a web-based graphical user interface with intrinsic queuing to control a distributed computational pipeline. RNA CoMPASS was evaluated by analyzing RNA-seq data sets from 45 B-cell samples. Twenty-two of these samples were derived from lymphoblastoid cell lines (LCLs) generated by the infection of naïve B-cells with the Epstein Barr virus (EBV), while another 23 samples were derived from Burkitt's lymphomas (BL), some of which arose in part through infection with EBV. Appropriately, RNA CoMPASS identified EBV in all LCLs and in a fraction of the BLs. Cluster analysis of the human transcriptome component of the RNA CoMPASS output clearly separated the BLs (which have a germinal center-like phenotype) from the LCLs (which have a blast-like phenotype) with evidence of activated MYC signaling and lower interferon and NF-kB signaling in the BLs. Together, this analysis illustrates the utility of RNA CoMPASS in the simultaneous analysis of transcriptome and metatranscriptome data. RNA CoMPASS is freely available at http://rnacompass.sourceforge.net/.

Dataset Information

Assessing Transcriptome Quality in Patch-Seq Datasets.

Publications

Assessing Transcriptome Quality in Patch-Seq Datasets.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets