Introduction

The 3D RNA-seq App takes transcript quantifications from Salmon (Patro et al., 2017) or Kallisto (Bray et al., 2016) as input for 3D analysis. The user manuals of command lines to generate quantifications can be found in:

For biologists, we recommend to use the Salmon/Kallisto tool in web-based graphical user interface Galaxy Europe (https://usegalaxy.eu/) or other Galaxy resources (https://galaxyproject.org/use/), in which users can perform transcript quantification and download results by “clicking mouse”.

Example data

Download link:

https://www.dropbox.com/s/k42kvxw9adrrcgp/Galaxy_example_data.zip?dl=0

Transcriptome:

  • A subset of AtRTD2 Arabidopsis transcriptome (Zhang et al, 2017) with 4679 transcripts from 1000 genes.

RNA-seq reads:

  • Two conditions, \(20^oC\) vs \(4^oC\).
  • Each has 3 biological replicates.
  • 150 bp paired-end reads.
Samples Temperature Bio-reps Read1 Read2
Sample1 20 Brep1 sample_01_1 sample_01_2
Sample2 20 Brep2 sample_02_1 sample_02_2
Sample3 20 Brep3 sample_03_1 sample_03_2
Sample4 4 Brep1 sample_04_1 sample_04_2
Sample5 4 Brep2 sample_05_1 sample_05_2
Sample6 4 Brep3 sample_06_1 sample_06_2

Registration of Galaxy Europe

https://usegalaxy.eu/


Note: The account needs email activation.

Galaxy user manual

Galaxy user manual can be found in: https://galaxyproject.org/learn/

Structure of Galaxy interface

Upload data to Galaxy

Galaxy user manual: https://galaxyproject.org/tutorials/upload/

Two types of input files are required for transcript quantification using Salmon:

  • RNA-seq reads in fasta/fastq format.
  • Transcript sequence file in fasta (.fa) format.



Salmon quantification

Input files:

  • Transcriptome sequence fasta file with estension “.fa”.
  • RNA-seq reads files of samples with estension “.fastq.gz” or “.fasta.gz”

Salmon documentation:

Im most cases, RNA-sesq data includes many samples and replicates. Galaxy allows users to build a list of data pairs from RNA-seq data. The user manual can be found in: Galaxy user manual can be found in: https://galaxyproject.org/tutorials/collections/







Kallisto quantification

Input files:

  • Transcriptome sequence fasta file with estension “.fa”.
  • RNA-seq reads files of samples with estension “.fastq.gz” (Kallisto in Galaxy does not take “.fasta.gz” format).

Kallisto documentation:



Prepare input files for 3D RNA-seq App

The 3D RNA-seq App reads transcript quantifications in “.tabular” files from Galaxy outputs.


References

Bray,N.L., Pimentel,H., Melsted,P., and Pachter,L. (2016) Near-optimal probabilistic RNA-seq quantification. Nat. Biotechnol., 34, 525–527.

Calixto,C.P.G., Guo,W., James,A.B., Tzioutziou,N.A., Entizne,J.C., Panter,P.E., Knight,H., Nimmo,H.G., Zhang,R., and Brown,J.W.S. (2018) Rapid and Dynamic Alternative Splicing Impacts the Arabidopsis Cold Response Transcriptome. Plant Cell, 30, 1424–1444.

Guo,W., Tzioutziou,N., Stephen,G., Milne,I., Calixto,C., Waugh,R., Brown,J.W., and Zhang,R. (2019) 3D RNA-seq - a powerful and flexible tool for rapid and accurate differential expression and alternative splicing analysis of RNA-seq data for biologists. bioRxiv, 656686. doi: https://doi.org/10.1101/656686.

Patro,R., Duggal,G., Love,M.I., Irizarry,R.A., and Kingsford,C. (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat. Methods, 14, 417–419.