The 10X single-cell sequencing technology is a technique that is used in the analysis of large cell numbers through single-cell expression profiling with the highest efficiency capture of up to 65 percent. The technology does allow not only the higher-throughput single-cell transcription but also the single-nuclei expression profiling. The technology has been beneficial in providing a comprehensive and scalable solution for both gene expression profiling and cell characterization of tens of thousands of cells. The 10X single cell sequencing technology operates through the cell ranger system which is a set of analysis of pipeline that aligns the chromium RNA-seq single cell output with reads to generate feature-barcode matrices to allow performance of the clustering and gene expression analysis.
I believe that the 10x single cell sequencing technology operates under the four pipelines/phases of the cell ranger system. The first pipeline is the cell ranger mkfastq which involves demultiplexes of the raw base call (BCL) files which are generated by illumine sequencers into FASTQ file. With additional useful traits that are only specific to 10X libraries, the raw base call is then wrapped around the Illumina’sbclfastq. The second pipeline is the call ranger count where the FASTQ file is taken from the call ranger mksfastq and then aligned, filtered, barcoded, and the UMI counted. During this phase, the chromium cellular barcodes are used to generate feature-bar code metrices which are applied to determine the cluster required in performing the gene expression analysis.
I also understand that barcoding plays a vital role in the 10X single-cell sequencing technology. When barcoding, the cells are encapsulated into nanoliter droplets which contains hydrogel beads (HBs) which bear the barcoding DNA primers. The third phase is the cell rangeraggr which involves the aggregation of output generated from the cell ranger count. The outputs are then normalized to the same sequencing depth, and then the feature-barcode matrices are recomputed and analyzed on a combined data generated. The last phase is the cell ranger reanalyze where the feature-barcode matrices produced from the previous phase are rerun through dimensional reduction, clustered, and expressed through gene algorithms.
10X Genomics. Single Cell Gene Expression. Retrieved from https://support.10xgenomics.com/single-cell-gene-expression/software/pipelines/latest/what-is-cell-ranger. (Accessed on 31st January 2019).
Wilson, Nicola K., et al. “Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations.” Cell stem cell 16.6 (2015): 712-724.
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