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Long-read sequencing captures the full transcript and thus minimizes many of issues in estimating isoform abundance, like ambiguous read mapping. For short-read RNA-Seq, there are multiple methods to detect alternative splicing that can be classified into three main groups: [119] [91] [120]
3' mRNA-seq methods are generally cheaper per sample than standard bulk RNA-seq methods. [2] [7] [8] [9] This is because of the lower sequencing depth required due to only the 3' end of mRNA molecules being sequenced instead of the whole length of entire transcripts. Read depths of between one million and five million reads are recommended in ...
RNA-Seq methodology has constantly improved, primarily through the development of DNA sequencing technologies to increase throughput, accuracy, and read length. [61] Since the first descriptions in 2006 and 2008, [ 40 ] [ 62 ] RNA-Seq has been rapidly adopted and overtook microarrays as the dominant transcriptomics technique in 2015.
At this step, sequencing reads whose quality have been improved are mapped to a reference genome using alignment tools like BWA [17] for short DNA sequence reads, minimap [18] for long read DNA sequences, and STAR [19] for RNA sequence reads. The purpose of mapping is to find the origin of any given read based on the reference sequence.
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]
Sequence coverage (or depth) is the number of unique reads that include a given nucleotide in the reconstructed sequence. [1] [2] Deep sequencing refers to the general concept of aiming for high number of unique reads of each region of a sequence. [3] Physical coverage, the cumulative length of reads or read pairs expressed as a multiple of ...
[2] [3] The mRNA sequence is determined by the sequence of genomic DNA. [4] In this context, the standard genetic code is referred to as translation table 1. [3] It can also be represented in a DNA codon table. The DNA codons in such tables occur on the sense DNA strand and are arranged in a 5 ′-to-3 ′ direction.
Superfast and accurate read aligners. Subread can be used to map both gDNA-seq and RNA-seq reads. Subjunc detects exon-exon junctions and maps RNA-seq reads. They employ a novel mapping paradigm named seed-and-vote. Yes Yes Yes Yes Free, GPL 3 Taipan De-novo assembler for Illumina reads Proprietary, freeware for academic and noncommercial use UGENE