TERAD: Extraction of transposable element composition from RADseq data

Transposable elements (TEs)—selfish DNA sequences that can move within the genome—comprise a large proportion of the genomes of many organisms. Although low-coverage whole genome sequencing can be used to survey TE composition, it is non-economical for species with large quantities of DNA. Here, we utilize restriction-site associated DNA sequencing (RADSeq) as an alternative method to survey TE composition.

In our paper (Chak et al, 2019), we demonstrate in silico that double digest restriction-site associated DNA sequencing (ddRADseq) markers contain the same TE compositions as whole genome assemblies across arthropods. Then, we show empirically using eight Synalpheus snapping shrimp species with large genomes that TE compositions from ddRADseq and low-coverage whole genome sequencing are comparable within and across species.

This bioinformatic pipeline, TERAD, is used to extract TE compositions from RADseq data.

IMPORTANT: Our pipeline used only one end of the pair-end reads to remove the bias from the rarity of EcoRI cut sites among known Arthropod TEs. We found that the cut frequency of EcoRI was lower than that of MspI (56% vs. 85%, respectively). Therefore, we analyzed only the EcoRI-ends of the paired-end reads to include only TEs that did not have an EcoRI restriction site. For your organism, you should decide which of you enzymes are the rare cutter and use that side of the pair-end for the analysis.

Installation

1. Download TERAD

Place contents in, for example, ~/Desktop/TERAD

2. Install RepeatMasker

Install RepeatMasker (http://www.repeatmasker.org/RMDownload.html) and its dependable programs including:

• RMBlast (http://www.repeatmasker.org/RMBlast.html)
• TRF (http://tandem.bu.edu/trf/trf.html).

Then run the RepeatMasker configuration script.

Our program has been tested using RepeatMasker,v 1.332 2017/04/17 with RMBlast 2.9.0-p1

3. Install cd-hit

https://github.com/weizhongli/cdhit

Our program has been tested using CD-HIT version 4.7 (built on Jan 25 2018).

4. Install R and packages

https://www.r-project.org/

Once R is installed, in terminal start R by typing R, then in the R console, type the following: install.packages("readr"); install.packages("plyr"); install.packages("fitdistrplus")

If you are installing on an HPC system, you may need to choose custom library location (for example see: https://www.osc.edu/resources/getting_started/howto/howto_install_local_r_packages)

5. Add paths for RepeatMasker and cd-hit to PATH.

To set path on, for example, a Mac

To edit the bash_profile: nano $HOME/.bash_profile add these lines at the end depending on the location where you have installed these programs:

export PATH=$PATH:/Users/solomon/Documents/programs/cdhit-master
export PATH=$PATH:/Users/solomon/Documents/programs/RepeatMasker

To save: type Control+ O, then Y, then Control+ X

To set path on, for example, HPC

nano .bashrc Add these lines:

PATH=/XXXX/XXXX/RepeatMasker:$PATH
PATH=/XXXX/XXXX/cdhit:$PATH

6. Now TERAD should be ready to go.

Test run:

(You may need to do: chmod +x TERAD)

cd ~/Desktop/TERAD
./TERAD test_file.fasta 4 ./arthro_ES_ND_PV_classified.fa none

How to run

Inputs are:

1. A file to search for TE in fasta format
2. The number of cores to use
3. The custom library to search for TE or "none"
4. Query species for RepeatMasker or "none"

** Use either inputs 3 or 4 and leave the other as “none.

Make sure your input file, TERAD, extract_cdhit2.R, and RAD_TE_summary.R in the same folder as well as your custom TE library in fasta format if you wish to use one.

Test run: ./TERAD test_file.fasta 4 ./arthro_ES_ND_PV_classified.fa none or ./TERAD test_file.fasta 4 none arthropods

Main output

The main output is test_file.fasta.cd.RAD_TE.summary2.

It is a .csv file that summarizes the proportions of RAD tags for each major TE subclass. .int and .ext indicate tags whether the restriction enzyme cut sites (e.g., EcoRI if we used EcoRI-ends of the paired-end ddRAD reads) were internal or external to the TE. We only analyzed TEs that don't have the EcoRI sites (i.e., the .ext proportions) to avoid the low cut frequency of EcoRI in known Arthropod TEs.

Column names	Notes
sample	Sample name
lib.depth	Total number of RAD tag
DNA	DNA transposons
RC	Helitron
LTR	LTR retrotransposon
LINE	Long interspersed nuclear elements
SINE	Short interspersed elements
RNA	RNA
UO	Unknown/Other
Sim	Simple repeat (Microsatellites)
Sat	Satellites
LC	Low complexity repeats
TE	Total TE
TE.int	Total TE where restriction sites are internal to the TE (inside the TE)
TE.ext	Total TE where restriction sites are external to the TE (outside the TE)
NoTE	RAD tags that have no TEs

Other output from different components of the pipeline

From cd-hit

• test_file.fasta.cd
• test_file.fasta.cd.clstr

From extract_cdhit2.R

• test_file.fasta.cd.summary2
• test_file.fasta.cd.summary1

From RepeatMasker

• test_file.fasta.cd.cat
• test_file.fasta.cd.out
• test_file.fasta.cd.tbl
• test_file.fasta.cd.masked
• test_file.fasta.cd.RM.progress

From RepeatProteinMask

• test_file.fasta.cd.masked.protM.progress
• test_file.fasta.cd.masked.annot
• test_file.fasta.cd.masked.masked
• test_file.fasta.cd.masked.rmsimple.cat.all

From RAD_TE_summary.R

• test_file.fasta.cd.RAD_TE.summary1
• test_file.fasta.cd.RAD_TE.summary2

Questions?

Email [email protected]