Bioinformatics pipeline for transcriptome sequencing analysis
How does RNA sequencing pipeline work?
The first protocol covers the standard pipeline on RNA-seq data that interrogates the transcriptome at the gene level, which is usually referred as differentially expressed gene (DEG) analysis. This pipeline starts from the raw sequence reads, and ends with a set of differentially expressed genes..
What is RNA-Seq analysis pipeline?
Abstract. RNA sequencing (RNA-seq) is a high throughput technology that provides unique insights into the transcriptome. It has a wide variety of applications in quantifying genes/isoforms, detecting non-coding RNA, alternative splicing, and splice junctions..
What is RNA-Seq analysis pipeline?
Abstract. RNA sequencing (RNA-seq) is a high throughput technology that provides unique insights into the transcriptome. It has a wide variety of applications in quantifying genes/isoforms, detecting non-coding RNA, alternative splicing, and splice junctions.Feb 13, 2019.
What is the best pipeline for RNA-Seq?
These results clearly show that counting and normalization methods are the most critical steps in the RNA-seq analysis process. Particularly, considering the above results, we concluded that the combination of Trimmomatic + RUM + HTSeq Union + TMM was the most precise and accurate pipeline..
What is the whole transcriptome sequencing pipeline?
Whole transcriptome sequencing enables the complete profiling of both mRNA and non-coding RNAs (including microRNAs, lncRNA, and circRNAs) in a biological sample under specific conditions by combining RNA-seq with rRNA depletion and small RNA-Seq..
Which technique is used for transcriptome analysis?
Currently, the two main transcriptomics techniques include DNA microarrays and RNA-Seq. Both techniques require RNA isolation through RNA extraction techniques, followed by its separation from other cellular components and enrichment of mRNA..
Abstract. RNA sequencing (RNA-seq) is a high throughput technology that provides unique insights into the transcriptome. It has a wide variety of applications in quantifying genes/isoforms, detecting non-coding RNA, alternative splicing, and splice junctions.
Currently, the two main transcriptomics techniques include DNA microarrays and RNA-Seq. Both techniques require RNA isolation through RNA extraction techniques, followed by its separation from other cellular components and enrichment of mRNA.
Summary of RNA-Seq. Within the organism, genes are transcribed and (in an eukaryotic organism) spliced to produce mature mRNA transcripts (red). The mRNA is extracted from the organism, fragmented and copied into stable ds-cDNA (blue). The ds-cDNA is sequenced using high-throughput, short-read sequencing methods.
The basic steps of an RNA-seq experiment involve RNA extraction, RNA fragmentation, cDNA generation, library amplification, and sequencing on an NGS platform to get strings of continuous sequence data in “reads”. The most common approach is short-read sequencing (read lengths ≤ 300 bp; RNA-seqlopedia).
The workflow includes three parts: (a) mapping sequencing reads to a reference genome or transcriptome; (b) quantifying expression levels of individual genes and transcripts; and (c) identifying specific genes and transcripts that are differentially expressed between samples.
The development of High Throughput Sequencing (HTS) for RNA profiling (RNA-seq) has shed light on the diversity of transcriptomes. While RNA-seq is becoming
We present the workflow using human transcriptome sequencing data from two biological replicates of the K562 cell line produced as part of the ENCODE3 project.
Mapping
STAR uses a suffix array approach to map reads to the genome and to the annotated splice junctions. Reads can be mapped both in a continuous way, i.e., in one block, or in a noncontinuous way, i.e., allowing gaps which can be considered as introns if long enough (see --alignIntronMin option below), in which case the read mapping is called a split-m.
Transcript and Gene Quantifications
RSEM uses reads mapped to the transcriptome to quantify the expression of transcripts and genes. It uses an expectation maximization approach to rescue multi-mapped reads based on the location of unique reads in the transcript, in an iterative way that stops when the error made is lower than a threshold. Preparing the RSEM reference files This need.
Transcriptome Reconstruction/Assembly
3.2.1 Cufflinks
What are the most commonly used analytical pipelines for transcriptome analysis?
Here, we compile and present a robust and most commonly used analytical pipelines covering entire spectrum of transcriptome analysis, including:
quality checks
aligning reads
differential gene/transcript expression analysis
cryptic splicing events discovery
and visualization.
What is a bioinformatics pipeline?
This pipeline quantifies annotated genes and transcripts, however the commands we provide are general enough to be easily extended to quantify both known and novel transcripts. Pipeline description. Schematic overview of the bioinformatics pipeline described in this protocol.
What is transcriptome profiling?
At present, transcriptome profiling with next-generation sequencing (NGS) data such as:
RNA-Seq and polyadenylation site sequencing (PAS-Seq)
is an indispensable method for performing quantitative analysis of gene or isoform expression ( Wang et al., 2017 ).
Which bioinformatics pipeline is used to process RNA-Seq reads?
Here we present a commonly used bioinformatics pipeline to process RNA-seq reads using STAR [ 3] for mapping sequences, Cufflinks [ 4] for transcript model reconstruction and RSEM [ 5] for transcript and gene quantifications ( see Fig. 1 ).
De novo transcriptome assembly is the de novo sequence assembly method of creating a transcriptome without the aid of a reference genome.
Bioinformatics pipeline for transcriptome sequencing analysis
In epitranscriptomic sequencing, most methods focus on either (1) enrichment and purification of the modified RNA molecules before running on the RNA sequencer, or (2) improving or modifying bioinformatics analysis pipelines to call the modification peaks. Most methods have been adapted and optimized for mRNA molecules, except for modified bisulfite sequencing for profiling 5-methylcytidine which was optimized for tRNAs and rRNAs.
Method for sequencing DNA
Single-molecule real-time (SMRT) sequencing is a parallelized single molecule DNA sequencing method. Single-molecule real-time sequencing utilizes a zero-mode waveguide (ZMW). A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA being incorporated by DNA polymerase. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
Set of all RNA molecules in one cell or a population of cells
The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The term transcriptome is a portmanteau of the words transcript and genome; it is associated with the process of transcript production during the biological process of transcription.
De novo transcriptome assembly is the de novo sequence assembly method of creating a transcriptome without the aid of a reference genome.
In epitranscriptomic sequencing
In epitranscriptomic sequencing, most methods focus on either (1) enrichment and purification of the modified RNA molecules before running on the RNA sequencer, or (2) improving or modifying bioinformatics analysis pipelines to call the modification peaks. Most methods have been adapted and optimized for mRNA molecules, except for modified bisulfite sequencing for profiling 5-methylcytidine which was optimized for tRNAs and rRNAs.
Method for sequencing DNA
Single-molecule real-time (SMRT) sequencing is a parallelized single molecule DNA sequencing method. Single-molecule real-time sequencing utilizes a zero-mode waveguide (ZMW). A single DNA polymerase enzyme is affixed at the bottom of a ZMW with a single molecule of DNA as a template. The ZMW is a structure that creates an illuminated observation volume that is small enough to observe only a single nucleotide of DNA being incorporated by DNA polymerase. Each of the four DNA bases is attached to one of four different fluorescent dyes. When a nucleotide is incorporated by the DNA polymerase, the fluorescent tag is cleaved off and diffuses out of the observation area of the ZMW where its fluorescence is no longer observable. A detector detects the fluorescent signal of the nucleotide incorporation, and the base call is made according to the corresponding fluorescence of the dye.
Set of all RNA molecules in one cell or a population of cells
The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The term transcriptome is a portmanteau of the words transcript and genome; it is associated with the process of transcript production during the biological process of transcription.
