Frequently asked question (FAQ) - Linlab-slu/TSSr GitHub Wiki

Q1: Could I use TSSr without BSgenome? issues#18

A1: No. In the current phase (2024-09-30), the current version (v0.99.1), TSSr is also highly dependent on and integrated with BSgenome and BSgenome packages for the species being processed.

Q2: Sometimes the exported TSS_correlation_plot_of_all_samples.pdf is too large to open. Is there any solution?

A2: Yes, you can use poppler to reduce the size of the PDF.

First, install poppler using conda:

conda install -c conda-forge poppler

Once installed, you can use the pdftoppm tool from poppler to convert the PDF into a more manageable format, such as PNG or JPEG, or optimize the PDF for easier viewing. For example:

# convert to png format
pdftoppm -png input.pdf output_prefix

# for example
pdftoppm -png TSS_correlation_plot_of_all_samples.pdf TSS_correlation_plot_of_all_samples

It will output a file with the name: TSS_correlation_plot_of_all_samples-1.png.

You can also convert the pdf format to other formats like: JEPG, TIFF and PPM (Portable Pixmap Format, default)

Also, you can set image resolution to 300 DPI by using -r option:

pdftoppm -png -r 300 input.pdf output_prefix

Q3: Why do we use splice-aware aligners for mapping CAGE data instead of non-splice-aware aligners like Bowtie/Bowtie2?

Because there are introns in UTR region. According to this paper:

Approximately 35% of human genes contain introns within the 5' untranslated region (UTR).

So if you are using a non-splice-aware aligner, like bowtie2, the reads could not find proper mapping region.

Q4: How can we determine the overall intron landscape within UTR regions of a species?

Q5: How to install the tools we need in the analysis process?

A5: We recommend you use miniforge. Here is a brief guide.

  1. download the miniforge installer.
wget -c https://github.com/conda-forge/miniforge/releases/latest/download/Miniforge3-Linux-x86_64.sh
  1. install it with bash.

Tips: When you been asked "yes" or "no", always choose yes. When haven't been asked, just press ENTER.

bash Miniforge3-Linux-x86_64.sh
  1. set channels

run these commands to set channles

conda config --add channels conda-forge
conda config --add channels bioconda
  1. set conda environment

Don't install all the tools in your base environment. Create a new one!

# create a env called tss
conda create -n tss
# install the tools
conda install star samtools fastq-dl

(TO BE REFINED) Q6：bigwig(.bw) format file generate failed but bedGraph format success, how to get .bw file?

Install the tools by conda：

conda install seqkit ucsc-bedgraphtobigwig

Generate genome name & length information with seqkit

seqkit fx2tab -n -l <genome.fasta> -o genome.name.length
bedGraphToBigWig <input.bedGraph> genome.name.length <output.bw>
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