More at https://www.igib.res.in/bdmg/ScientistRecruitmentAdvt2023.pdf

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.

ADVERTISEMENT NO: NITR/SR/CH-BIF/2014/30

Applications are invited on prescribed format for the following assignment in a purely time bound research project undertaken in the Department of Biotechnology & Medical Engineering of the Institute.

1. Name of the Temporary Post : Senior Research Fellow-01
2. Name of the Research Project: “ Bioinformatics Infrastructure Facility (BIF)”
3. Name of the Sponsoring Agency: DBT, Government of India, 4 Tenure of the Project : 12th Five year Plan
5 Tenure of the Assignment : 01 year [Likely to be extended for 04 more years]
6 Job Description : BIF Maintenance and Active Research in Bioinformatics
7. Consolidated monthly compensation / Fellowship: Rs.18,000/- P.M.

8. Essential Qualifications and experience: B.Tech with valid GATE Score or M.Tech degree in Biotechnology/Bioinformatics/Computer Science/Computational Biology
9. Desirable Qualifications/ Experiences: Experience of Programming in PERL,R, Python, Unix and Visual Studio + Knowledge in NGS data analysis work flows ,WGS and statistical packages such as CRAN-R,MATLAB etc.

10. Accommodation : Bachelor accommodation in the Institute may be provided subject to availability.
11. For technical information on the project, the candidate may contact the Principal Investigator at the following address:

Name : Prof. Mukesh K Gupta
Address : Dept. of Biotechnology & Medical Engineering,
N.I.T.Rourkela-769 008
Telephone No : 0661-2462294
E-mail : guptam@nitrkl.ac.in

Eligible persons may apply in the prescribed format (available in the Institute Website)affixed with coloured photographs to be submitted in duplicate along with photo copies of relevant certificates, grade/ mark sheets, publications etc., to Asst. Registrar, SRICCE,
National Institute of Technology, Rourkela–769 008 before 22.08.2014. The cover should be super- scribed clearly the post applied for & Name of the Project.

Mere possession of minimum qualification does not guarantee invitation to the interview.
Candidates will be short listed based on merit and need of the project.

Advertisement:

http://www.nitrkl.ac.in/IntraWeb/Jobs_Tenders/Jobs/ProjectFellowship/2014/141707192838_1.pdf

More at https://leylab.com/

Studentship and Traineeship in Bioinformatics

Applications are invited on plain paper from suitable candidates for Studentship and Traineeship (One each) at Bioinformatics Sub-Center as detailed below:

1. Studentship: Studentship is for those who have completed M. Sc. Degrees in Life Science.

Number of seats : One

Duration : Six months

Eligibility : Passed M.Sc. degree in Life Sciences.

Fellowship : Rs. 5000/- (Five thousand only) per month

2. Traineeship: Traineeship is for those who have completed M. Sc. Degrees in Life Science/Registered Ph. D. student in Life Sciences.

Number of seats : One

Duration : Six months

Eligibility : Passed M.Sc. degree in Life Sciences/ Registered Ph. D. student in Life Sciences

Fellowship : Rs. 5000/- (Five thousand only) per month

Preferences will be given to person who has experience in Bioinformatics and Computer
sciences. The application along with detailed bio-data should reach the undersigned, on or before 25th August 2014. Both, the studentship and the traineeship are temporary, will be discontinued after the six months from the date of Joining. It may be discontinued in-between without any notice, if the work is not found satisfactory.

Advertisement www.bioinfobubpl.nic.in/Advertisement_st.pdf

http://www.i-programmer.info/news/181-algorithms/4537-a-new-dna-sequence-search-compressive-genomics.html

http://en.wikipedia.org/wiki/Compression_of_Genomic_Re-Sequencing_Data

http://www.nature.com/nbt/journal/v30/n7/full/nbt.2241.html

http://bioinformatics.oxfordjournals.org/content/29/13/i283.full

http://groups.csail.mit.edu/cb/cast/

The vital metaphase stage of cell division, when the sister chromatids migrated to the centre and lined up in a row, and pulled apart using attached microtubules in such a way that half the DNA ends up in each daughter cell. However, before the mitotic spindle‐mediated movement gets start and pulled DNA apart, the chromosomes are free to undergo recombination which involves the exchange of genetic material either between multiple chromosomes or between different regions of the same chromosome.

During recombination, the precise breakage of each strand, exchange between the strands, and sealing of the resulting recombined molecules happens. The “chromosomal breakpoints” refers to these places where they break. Mostly, this process occurs with a high degree of accuracy at high frequency in both eukaryotic and prokaryotic cells. But occasionally this “break and sealing/ break and reattach” process goes wrong and the reattachment happens in the wrong place which usually create disaster (with few exceptions).These chromosome disaster or abnormalities involve the gain, loss or rearrangement of visible amounts of genetic material during cell division. These abnormalities are of two type, the first one is numerical abnormalities  where severe disorders are caused by the loss or gain of whole chromosomes, which affect the copy number of hundreds or even thousands of genes. The second are structural abnormalities which can be unbalanced or balanced. The former are similar to numerical abnormalities in that genetic material is either gained or lost. The natural defects in chromosome segregation are linked to cancer and several genetic diseases (http://en.wikipedia.org/wiki/List_of_genetic_disorders). Therefore, the enzymes involved in regulating cell division are still the attractive drug targets for many diseases.

Apart from certain chromosome abnormalities, these “crossing over” of segments of maternal and paternal chromosomes to form hybrid chromosomes have some evolutionary importance and considered as a driver of genetic variation. Moreover, the chromosome breakage in evolution is considered to be non-random in nature(http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.0020014). In addition the study of breakpoint regions and non-breakpoint (stable) regions of chromosomes indicates both the regions evolved in distinctly different ways ( http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2675965/). These breakage may lead to genetic diseases or participate to chromosomal rearranmgnets and contributed in development of new species.

I will try to explain the genome hotspots/Evolutionary Breakpoint Regions(EBRs)/fragile regions/weak fragments/  in my next blog.

Software for recombination detection:

RAT http://cbr.jic.ac.uk/dicks/software/RAT/

Breakpointer https://github.com/ruping/Breakpointer

DRP http://web.cbio.uct.ac.za/~darren/rdp.html

RB-finder http://www.ncbi.nlm.nih.gov/pubmed/18707535

LDhat2.0 http://ldhat.sourceforge.net/LDhat2.0/instructions.shtml

Reference:

http://www.nature.com/scitable/topicpage/genetic-recombination-514#

Image: Wikipedia , sciencelearn.org.nz

Recommended Articles:

http://www.friendshipcircle.org/blog/2012/05/22/13-chromosomal-disorders-youve-never-heard-of/

http://web.udl.es/usuaris/e4650869/docencia/segoncicle/genclin98/recursos_classe_%28pdf%29/revisionsPDF/chromosyndromes.pdf

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2775595/table/T2/

http://learn.genetics.utah.edu/content/disorders/chromosomal/

http://www.ncert.nic.in/html/learning_basket/biology/cc&cd.pdf

Abstract: Strand NGS includes comprehensive workflows for DNA-Seq, RNA-Seq, Small RNA-Seq, ChIP-Seq, MeDIP-Seq, and Methyl-Seq analysis. Each workflow includes a quality assessment and filter section, followed by a workflow-specific analysis section. The pipeline functionality in Strand NGS allows users to execute a sequence of analysis steps with specific parameters - all without any manual intervention. This simplifies the analysis in large scale sequencing projects where every sample needs to be processed identically.

In this webinar we will discuss the pre-packaged pipelines present in Strand NGS. The packaged pipelines have well-chosen default parameters and are suitable for users analyzing data for the first time in the tool. We will also show how advanced users can customize pipelines and share them with other Strand NGS users. Finally, we will show a brief glimpse of an elaborate pipeline that aligns reads, filters poor-quality matches, computes coverage metrics, identifies variants, checks for sample cross-contamination, and emails quality reports - all from within Strand NGS.

Speaker: Dr. Vamsi Veeramachaneni, Vice President - Bioinformatics, Strand Life Sciences

Details: Session 1: 2:30 PM IST, Session 2 : 10:30 PM IST
Register here: http://www.strand-ngs.com/webinar_registration

Walk in interview for guest faculty in Pondicherry University, India. For more information please visit http://www.bicpu.edu.in/bioinfor140814.pdf

CGHub
https://cghub.ucsc.edu/
Comprehensive data repository; huge data size

EGA
https://www.ebi.ac.uk/ega/
Comprehensive data repository; huge data size

COSMIC
http://cancer.sanger.ac.uk
Largest somatic mutation database; genome sequencing paper curation

CPRG
http://www.broadinstitute.org/software/cprg
Interface for cancer program resources

GDAC
http://gdac.broadinstitute.org/
Data analysis; automatic pipelines; user-friendly reports

SNP500Cancer
http://snp500cancer.nci.nih.gov
Sequence and genotype verification of SNPs

canEvolve
www.canevolve.org/
Comprehensive analysis of tumor profile; Data from 90 studies involving more than 10,000 patients

MethyCancer
http://methycancer.psych.ac.cn
Relationship among DNA methylation, gene expression and cancer

SomamiR
http://compbio.uthsc.edu/SomamiR/
Correlation between somatic mutation and microRNA; genome-wide displaying

cBioPortal
http://www.cbioportal.org/public-portal/
Graphical summaries; gene alteration; processed data; visualization

UCSC Cancer Genomics Browser
https://genome-cancer.soe.ucsc.edu/
Clinical information; gene expression; copy number variation; visualization

CGWB
https://cgwb.nci.nih.gov/
Visualization; gene mutation and variation; automated analysis pipeline

GDSC
http://www.cancerrxgene.org
Drug sensitivity information; drug response information

canSAR
https://cansar.icr.ac.uk/
Multidisciplinary information; drug discovery

NONCODE
http://www.noncode.org/ ncRNAs;
lncRNAs; up-to-date and comprehensive resource