What is cufflinks RNA-Seq?

Cufflinks consist of a suite of tools for differential gene expression analysis of RNA-seq data. It assembles aligned reads in a set of transcripts and estimates the relative abundances.

Which sequencing platform is the best for RNA-Seq?

Illumina HiSeq platform
Currently, the Illumina HiSeq platform is the most commonly applied next-generation sequencing technology for RNA-Seq and has set the standard for NGS sequencing. The platform has two flow cells, each providing eight separate lanes for sequencing reactions to occur.

Which RNA is used in sequencing?

In the standard Iso-Seq protocol, high-quality RNA is converted to full-length cDNA for sequencing using a template-switching reverse transcriptase38,39. The resulting cDNAs are PCR amplified and used as the input for PacBio single-molecule, real-time (SMRT) library preparation.

What does RNA sequencing detect?

Not limited to genomic sequences – unlike hybridization-based approaches, which may require species-specific probes, RNA-seq can detect transcripts from organisms with previously undetermined genomic sequences. This makes it fundamentally superior for the detection of novel transcripts, SNPs or other alterations.

What is HT seq?

HTSeq is a Python package that calculates the number of mapped reads to each gene.

What are the steps in RNA sequencing?

A typical RNA-seq experiment consists of the following steps:

1. Design Experiment. Set up the experiment to address your questions.
2. RNA Preparation. Isolate and purify input RNA.
3. Prepare Libraries. Convert the RNA to cDNA; add sequencing adapters.
4. Sequence. Sequence cDNAs using a sequencing platform.
5. Analysis.

How much RNA do you need for RNA-Seq?

The standard protocol for library construction requires between 100 ng and 1 μg of total RNA. There are kits available for ultra-low RNA input that start with as little is 10 pg-10ng of RNA; however, the reproducibility increases considerably when starting with 1-2 ng.

Why is RNA-seq better?

The advantage of RNA-Seq over microarrays is that it provides an unbiased insight into all transcripts (Zhao et al., 2014). Thus, RNA-Seq is generally reliable for accurately measuring gene expression level changes.

What is deep RNA sequencing?

Deep sequencing refers to sequencing a genomic region multiple times, sometimes hundreds or even thousands of times. This next-generation sequencing (NGS) approach allows researchers to detect rare clonal types, cells, or microbes comprising as little as 1% of the original sample.

Why do we sequence RNA?

Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments.

How is cufflinks used in RNA Seq workflow?

The Cufflinks RNA-Seq workflow. The Cufflinks suite of tools can be used to perform a number of different types of analyses for RNA-Seq experiments. The Cufflinks suite includes a number of different programs that work together to perform these analyses.

What can cufflinks tell you about a gene?

Cuffdiff is a highly accurate tool for performing these comparisons, and can tell you not only which genes are up- or down-regulated between two or more conditions, but also which genes are differentially spliced or are undergoing other types of isoform-level regulation.

Which is the best tool for RNA Seq analysis?

TopHat and Cufflinks do not address all applications of RNA-seq, nor are they the only tools for RNA-seq analysis. In particular, TopHat and Cufflinks require a sequenced genome (see below for references to tools that can be used without a reference genome).

How are cufflinks used to normalize differential expression?

With this option, Cufflinks normalizes by the upper quartile of the number of fragments mapping to individual loci instead of the total number of sequenced fragments. This can improve robustness of differential expression calls for less abundant genes and transcripts.