Link @ http://www.bioinf.uni-freiburg.de/

RNAs, especially non-coding RNAs (such as microRNA, long ncRNAs) are recently identified to be very abundant in mammalian organisms and play some key roles in gene expression regulation, gene silencing, and also implicated in disease progression, stem cell pluripotency etc. Current research activities of our lab include analysis of expression pattern of ncRNAs by microarray and next-gen sequencing data and understanding the role of miRNAs or other regulatory RNAs in various diseases, especially cancer and validation by reporter assays (renilla/luciferase) and other experimental tools.

More @ http://vvekslab.in/index.html

http://www.chekulaevalab.org/

It takes FASTA DNA sequence as input, and write GFF3 as output. It uses the new NHMMER tool that comes with HMMER 3.1 for HMM searching in RNA:DNA style. NHMMER binaries for 64-bit Linux and Mac OS X are included and will be auto-detected. Multithreading is supported and one can expect roughly linear speed-ups with more CPUs. 

Address of the bookmark: https://github.com/tseemann/barrnap

http://lin-group.cn/server/iRNAD/

Address of the bookmark: http://lin-group.cn/server/iRNAD/

Address of the bookmark: http://huanglab.phys.hust.edu.cn/hnadock/

CPAT - https://github.com/liguowang/cpat, http://lilab.research.bcm.edu/ (web server)

CPC2 - https://github.com/gao-lab/CPC2_standalone, http://cpc2.gao-lab.org/ (web server)

Infernal - http://eddylab.org/infernal/, https://github.com/EddyRivasLab/infernal

NCBI RefSeq - https://www.ncbi.nlm.nih.gov/refseq/

Rfam - http://rfam.xfam.org/, https://docs.rfam.org/en/latest/index.html

SILVA - https://www.arb-silva.de/

RNAmmer - http://www.cbs.dtu.dk/services/RNAmmer/ (web server, standalone download link)

Why Normalize RNA-Seq Data?

Normalization is a crucial step in RNA-Seq analysis for the following reasons:

• Sequencing depth: Different RNA-Seq experiments produce varying numbers of reads, making direct comparisons between samples misleading.

• Gene length: Longer genes inherently generate more reads, irrespective of their actual expression level.

• Bias reduction: Normalization mitigates technical biases, enabling meaningful biological interpretation.

TPM (Transcripts Per Million)

TPM measures the proportion of reads mapped to a transcript, normalized by transcript length and sequencing depth. It is calculated as:

Key Features:

1. Proportionality: TPM values sum to 1,000,000 across all transcripts in a sample, making it easier to compare between samples.

2. Intuitive interpretation: TPM values directly represent the abundance of transcripts in a sample.

3. Preferred for comparisons: TPM facilitates between-sample comparisons better than FPKM.

FPKM (Fragments Per Kilobase Million)

FPKM normalizes read counts by transcript length and sequencing depth, but without enforcing proportionality like TPM. It is defined as:

Key Features:

1. Historical significance: FPKM was one of the first normalization methods used for RNA-Seq.

2. Single-end vs. paired-end: In paired-end sequencing, FPKM becomes RPKM (Reads Per Kilobase Million).

3. Limited utility: FPKM values are not as robust as TPM for cross-sample comparisons due to lack of proportionality.

CPM (Counts Per Million)

CPM normalizes raw read counts by sequencing depth, without considering gene length. It is expressed as:

Key Features:

1. Simplicity: CPM is straightforward and computationally less intensive.

2. Application: Suitable for non-length-dependent analyses, such as comparing total expression levels or differential expression analysis.

3. Gene length agnostic: CPM does not correct for gene length, making it less ideal for measuring expression levels.

When to Use Each Method

• TPM: Best for comparing expression levels between samples, especially when transcript length and sequencing depth vary.

• FPKM: Useful for historical consistency but generally replaced by TPM.

• CPM: Ideal for differential expression analysis when gene length normalization is unnecessary.

Conclusion

Choosing the right normalization method depends on the specific objectives of your RNA-Seq analysis. TPM’s proportionality and robustness make it the preferred choice for most applications, while CPM serves well for differential expression studies. Although FPKM paved the way for RNA-Seq normalization, it has largely been supplanted by TPM in modern workflows. Understanding these methods and their nuances ensures accurate and meaningful interpretations of RNA-Seq data.

References:

1. Li, B., & Dewey, C. N. (2011). RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics.

2. Trapnell, C., et al. (2010). Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnology.

3. Law, C. W., et al. (2014). voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biology.