• Generate a cell x gene or cell x transcript equivalence class count matrix
• Perform RNA velocity and single-nuclei RNA-seq analsis
• Quantify data from numerous technologies such as 10x, inDrops, and Dropseq.
• Customize workflows for new technologies and protocols.
• Process feature barcoding data such as CITE-seq, REAP-seq, MULTI-seq, Clicktags, and Perturb-seq.
• Obtain QC reports from single-cell RNA-seq data

The kallisto | bustools workflow is described in:

Páll Melsted*, A. Sina Booeshaghi*, Lauren Liu, Fan Gao, Lambda Lu, Kyung Hoi (Joseph) Min, Eduardo da Veiga Beltrame, Kristján Eldjárn Hjörleifsson, Jase Gehring & Lior Pachter† Modular and efficient pre-processing of single-cell RNA-seq, Nature Biotechnology (2021).

Documentation and tutorials for the kallisto bustools workflow are available at http://pachterlab.github.io/kallistobustools. 

https://www.nature.com/articles/s41587-021-00870-2

Address of the bookmark: https://pachterlab.github.io/kallistobustools/

Tool link:

http://www.cs.cmu.edu/~ckingsf/software/sailfish/

Address of the bookmark: http://www.genengnews.com/gen-news-highlights/lightweight-algorithms-sail-through-rna-sequencing-data/81249765/

ORLANDO, USA, Oct 17, 2017/ PRNewswire/

Strand NGS now supports large scale RNA- and small-RNA-Seq and Unique Molecular Identifiers (UMIs) for DNA-, RNA-, and small-RNA-Seq.

Strand Life Sciences announced the latest version release of its bioinformatics flagship product, Strand NGS, at the Annual Meeting of the American Society of Human Genetics today. Two major themes in Strand NGS v3.1 address recent challenges in next generation sequencing (NGS).

The first theme is large-scale RNA-Seq data analysis. Current cross-cohort RNA- and small-RNA-Seq studies span tens of replicates and batches across hundreds of samples, sometimes conducted across several different institutions. For such studies, Strand NGS v3.1 includes confounding variable analysis to eliminate technical effects, including batch effects; the t-SNE plot; profile and heat-map plots of gene-body coverage; and several other notable visual enhancements.

The second new feature is support for Unique Molecular Identifiers, or UMIs, for DNA-, RNA- and small-RNA-Seq. UMI support in Strand NGS is end-to-end, spanning alignment to variant calling in DNA-Seq, and alignment to quantification in RNA- and small-RNA-Seq. The Bioo Scientific, Qiagen, and Rubicon UMI protocols are natively supported, and an intuitive interface allows the specification of custom UMI protocols.

“For liquid biopsies and low-grade FFPE samples, UMI support in DNA-Seq enables the detection of somatic variants at low concentrations. In RNA-Seq, large-scale and UMI support can be used in single-cell-based studies that reveal tumor-cell heterogeneity, even at low concentrations”, says Dr. Vamsi Veeramachaneni, Chief Scientific Officer, Strand Life Sciences.

“At Strand, we are continuously working towards improving the accuracy and efficiency of NGS data analysis. Customers can look forward to Strand NGS becoming available on the cloud in the near future”, says Dr. Ramesh Hariharan, Chief Executive Officer, Strand Life Sciences.

Visit Strand Life Sciences at ASHG booth #1017 to know more about Strand NGS v3.1 and other products and service offerings from Strand Life Sciences. Click here to access detailed agenda and v3.1 release notes.

About Strand Life Sciences

Strand Life Sciences is a premier life science informatics innovation company. Founded in 2000, Strand is a leader in technology innovations for healthcare using genomics. By enhancing sequence-based diagnostics and clinical genomic data interpretation using a strong foundation of computational, scientific, and medical expertise, Strand is bringing individualized medicine to the world. To know more, visit www.strandls.com

Address of the bookmark: http://www.strand-ngs.com/strand-announce-strandngss-v31

Address of the bookmark: https://ccb.jhu.edu/software/stringtie/

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.

Address of the bookmark: https://bioconductor.org/packages/3.7/bioc/vignettes/dupRadar/inst/doc/dupRadar.html

The most common newbie questions are:

Should I try to use these free open source programs?  Why are we not trying GUI software for computational analysis? Should I use commercial bioinformatics programs/software?”

1. Let’s be open

We generally think free and cheap are useless. But this concept is not applicable when we discuss open source software. Mostly, the bioinformatics software is developed by highly competitive biological programmers who believe in open sharing of knowledge. They come under Open Bioinformatics Foundation or O|B|F which is a non-profit, volunteer run organization focused on supporting open source programming in bioinformatics. The best part about open source tools/software is that they’re free to download the source code and read exactly what the program does. If you are so inclined, you can view all of the parts of the program and see the logical flow of the pipeline. In addition, open source makes an excellent learning tool for any beginning bioinformatician. Moreover, you can modify existing open source programs to deal with cutting-edge problems or to customize your pipeline. Apart from your computational and analysis work, most of the reviewer also prefers the open source based results so that they can validate the results if validation required.

2. Code headache

As a bioinformatician you are supposed to know the basics of programming languages, and if you are not good at it, then please learn it as soon as possible because you are not a bio-analyst but biological programmers. The open source programs usually lack dedicated service and support teams (often because they were the product of an overworked doc/postdoc!) so you are responsible for troubleshooting your own errors most of the time. We commonly receive the HELP email to support and assist to setup the pipeline; you can also find this kind of request on any QA forum. I personally believe this coding horror brings the biggest downside of open-source programs; where you need some programming skills in order to implement the program in your pipeline. But, if you are not able to fix the pipeline and modify the open source code according to your requirements them you should re-think on your bioinformatician name tag!!!

3. Dive into the codes

Some of the biologist turn bioinformatician says “if you can do the same thing with commercial software then why to get migraine with weird codes”, well this statement looks to me that guys are keen to learn swimming but still don’t like to get wet. If you are still using paid software and doing your work by customer support and clicking some of the well-designed GUI button then perhaps you are not interested in learning and trying new and challenging bioinformatics works. You are missing the basic flavour of bioinformatics. Let’s dive into the coding world, I am sure your will enjoy it. I recommend your to swim freely in code’s sea, and enjoy the journey; do not merely watch it from the outside.  

4. Paid does not mean better

The bioinformatics company which are specializes in bioinformatics solutions develop well designed/packed, user friendly software by using a large number of specialised scientist, programmers and support staff. They also provide good services to accomplice your biological analysis work. This means that if you hit a ‘snag’ with your data, help is likely only a phone call away! These companies price their products competitively against the cost of a dedicated bioinformatician. You may be able to afford the program, but not the additional staff! Additionally, most of the functionality that you need in your analysis is already coded into the program. Need to plot a graph? Just click this button right here. It is that easy. But, as a bioinformatician this is not generally well encouraged approach in biological analysis work, because the software is not available to everyone and your data can’t be validated. Moreover, there is very less chances that anyone will repeat your work or love to do similar kind of research (because not all the labs in the world are rich like yours).

5. Take a caution

In biological analysis work, in which you deal GB/TB of data are having maximum chances of getting errors, so please be careful and always cross check your data before coming to any conclusion. Even an error in two line code can alter your entire analysis and display weird results. Some of the scientist blindly believes on commercial software, which is entirely wrong. Using proprietary tools does not absolve you of the need to actually read and research the type of analysis that you are doing. This is particularly true in the case of genome assembly and annotation.

At the end, I would like to tell only one think that open source solutions allows you to do more cutting edge analysis than the commercial tools. So let’s go for it.

Disclaimer:

This is my personal view. I have nothing to do with any company or open source community. The views expressed on these pages are mine alone and not those of my current/past employers. I do reserve the right to remove comments left by spammers or off-topic comments.

For knowledge about Linux and their importance amongst bioinformatician plesae read this article "An introduction to Linux for bioinformatics" by Paul Stothard.

Linux cheat sheet at http://bioinformaticsonline.com/file/view/87/linux-cheat-sheet

Please browse for futher useful linux pages on right hand side ...