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.

No.NITW/BT/2014/adhoc

APPLICATIONS ARE INVITED FOR THE APPOINTMENT OF ADHOC FACULTY ON CONTRACT BASIS IN THE DEAPARTMENT OF BIOTECHNOLOGY

Period of Contract: Initially the appointment is for one semester i.e., from July 2014 up to December 2014 only.

Essential Qualifications:

i) B. Tech or equivalent in Biotechnology/ Industrial Biotechnology/ Biochemical Engineering / Chemical Engg. Or M. Sc in Microbiology/ Botany/ Zoology/ Biochemistry/Biotechnology and ii) M. Tech or equivalent in Biotechnology/Industrial Biotechnology/Bioinformatics

Or

Integrated M. Tech in Biotechnology/Industrial Biotechnology/ Bioinformatics

Candidates must possess First class (60% aggregate marks or 6.5 CGPA) at B. Tech/ M. Sc and M. Tech.

Desirable: Ph. D Pay Package: All selected candidates shall be eligible for a consolidated pay of Rs.30, 000/- per month. Candidates with Ph. D shall be eligible for an additional amount of Rs.5, 000/- per month.

How to apply : Applications on plain paper with attested photocopies of certificate and bio data along with justification for eligibility should reach to the Head, Department of Biotechnology, National Institute of Technology, Warangal AP 506004 in the form of soft or hard copy on or before 21st June 2014 email : biotech_hod@nitw.ac.in

Intimation: No separate call letters will be sent to the candidates. All the eligible candidates will be notified in the institute web site on 23rd June 2014. All the eligible candidates are requested to report for the interview to the Head, Department of Biotechnology at 9:00 AM on 27th June 2014

Joining: Selected candidates will be informed and they are expected to join immediately.

Advertisement:

http://www.nitw.ac.in/nitw/announcements/2014/Bio-Adhoc%20Advt.%20May-2014.pdf

Shell history search command

Type history at a shell prompt:
$ history

It will display the list of all used commandline history with an serial number.

To search particular command, enter:
$ history | grep command-name
$ history | egrep -i 'scp|ssh|ftp'
Emacs Line-Edit Mode Command History Searching

To get previous command containing string, hit [CTRL]+[r] followed by search string:

(reverse-i-search):

To get previous command, hit [CTRL]+[p]. You can also use up arrow key.

CTRL-p

To get next command, hit [CTRL]+[n]. You can also use down arrow key.

CTRL-n

fc command

Apart from hostory command there are fc command to extract the command from history. The fc stands for either "find command" or "fix command.

For example list last 10 command, enter:
$ fc -l 10
To list commands 130 through 150, enter:
$ fc -l 130 150
To list all commands since the last command beginning with ssh, enter:
$ fc -l ssh
You can edit commands 1 through 5 using vi text editor, enter:
$ fc -e vi 1 5

Delete command history

The -c option causes the history list to be cleared by deleting all of the entries:
$ history -c

“As we roll out genomic medicine we are fighting against this society-wide misconception that having the bad gene means you’re going to get the disease. That’s only true in a very few cases.”

Source:Today Health

Address of the bookmark: http://www.today.com/health/should-you-get-sequenced-not-all-bad-genes-predict-disease-8C11017154

Address of the bookmark: http://infoplatter.wordpress.com/2014/04/06/bioinformaticians-pocket-reference/

Title: Comparison of Short Read De Novo Alignment Algorithms 

Author: Nikhil Gopal

Address of the bookmark: http://biochem218.stanford.edu/Projects%202011/Gopal%202011.pdf

Link:

http://www.nytimes.com/2013/11/21/science/two-surprises-in-dna-of-boy-found-buried-in-siberia.html?_r=0

http://www.nature.com/nature/journal/vaop/ncurrent/full/nature12736.html

The work will focus on the functional studies of small RNAs by using next-generation sequencing approaches.

Candidates should hold a Ph.D. degree and have strong background in bioinformatics.
The University of Nice Sophia-Antipolis provides a wide range of facilities and training essential for biomedical research.
Interested applicants should send a PDF with a cover letter stating research interests and qualifications, an updated CV, a summary of previous research experience and contact information for two references to Michele Trabucchi ( mtrabucchi@unice.fr )