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RNA-Seq (named as an abbreviation of RNA sequencing) is a technique that uses next-generation sequencing to reveal the presence and quantity of RNA molecules in a biological sample, providing a snapshot of gene expression in the sample, also known as transcriptome.
The earliest RNA-Seq work was published in 2006 with one hundred thousand transcripts sequenced using 454 technology. [40] This was sufficient coverage to quantify relative transcript abundance. RNA-Seq began to increase in popularity after 2008 when new Solexa/Illumina technologies allowed one billion transcript sequences to be recorded.
One type of sequencing method can be used in preference to another depending on the type of the sample, for a genomic sample assembly-based methods is used; for a metagenomic sample it is preferable to use read-based methods. [10] Metagenomic sequencing methods have provided better results than genomics, due to these present fewer false negatives.
snRNA-seq uses isolated nuclei instead of the entire cells to profile gene expression. That is to say, scRNA-seq measures both cytoplasmic and nuclear transcripts, while snRNA-seq mainly measures nuclear transcripts (though some transcripts might be attached to the rough endoplasmic reticulum and partially preserved in nuclear preps). [7]
RNA Seq Experiment. The single-cell RNA-seq technique converts a population of RNAs to a library of cDNA fragments. These fragments are sequenced by high-throughput next generation sequencing techniques and the reads are mapped back to the reference genome, providing a count of the number of reads associated with each gene. [13]
Since then, many advances in Next Generation Sequencing methods have been made. [6] Transcriptomes are now able to be routinely developed due to advances in these methods and new technologies such as microarrays and RNA-Seq. Both methods require computed imaging as well as high reads and statistical analysis. [5]
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An RNA spike-in is an RNA transcript of known sequence and quantity used to calibrate measurements in RNA hybridization assays, such as DNA microarray experiments, RT-qPCR, and RNA-Seq. [ 1 ] A spike-in is designed to bind to a DNA molecule with a matching sequence , known as a control probe .