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When looking at groups of features across samples, FPKM is converted to transcripts per million (TPM) by dividing each FPKM by the sum of FPKMs within a sample. [ 91 ] [ 92 ] [ 93 ] Total sample RNA output: Because the same amount of RNA is extracted from each sample, samples with more total RNA will have less RNA per gene.
Sequencing technologies vary in the length of reads produced. Reads of length 20-40 base pairs (bp) are referred to as ultra-short. [2] Typical sequencers produce read lengths in the range of 100-500 bp. [3] However, Pacific Biosciences platforms produce read lengths of approximately 1500 bp. [4] Read length is a factor which can affect the results of biological studies. [5]
It is already adapted to align long reads (third-generation sequencing technologies) and can reach speeds of 45 million paired reads per hour per processor. [49] Subjunc [44] is a specialized version of Subread. It uses all mappable regions in an RNA-seq read to discover exons and exon-exon junctions.
The average coverage for a whole genome can be calculated from the length of the original genome (G), the number of reads (N), and the average read length (L) as /. For example, a hypothetical genome with 2,000 base pairs reconstructed from 8 reads with an average length of 500 nucleotides will have 2× redundancy.
A human transcriptome could be accurately captured using RNA-Seq with 30 million 100 bp sequences per sample. [85] [86] This example would require approximately 1.8 gigabytes of disk space per sample when stored in a compressed fastq format. Processed count data for each gene would be much smaller, equivalent to processed microarray intensities.
While single-read accuracy is 87%, consensus accuracy has been demonstrated at 99.999% with multi-kilobase read lengths. [ 37 ] [ 38 ] In 2015, Pacific Biosciences released a new sequencing instrument called the Sequel System, which increases capacity approximately 6.5-fold.
In bioinformatics, sequence assembly refers to aligning and merging fragments from a longer DNA sequence in order to reconstruct the original sequence. [1] This is needed as DNA sequencing technology might not be able to 'read' whole genomes in one go, but rather reads small pieces of between 20 and 30,000 bases, depending on the technology used. [1]
Fast gapped aligner and reference-guided assembler. Aligns reads using a banded Smith-Waterman algorithm seeded by results from a k-mer hashing scheme. Supports reads ranging in size from very short to very long. Yes MPscan Fast aligner based on a filtration strategy (no indexing, use q-grams and Backward Nondeterministic DAWG Matching) [47] 2009