Inventory of transposon-related gene isoforms

In brief, ParasiTE is a tool aiming to:

1. Identify TEs located in exonic or intronic regions of genes.
2. Detect TE sequences that are co-transcribed with gene mRNA (TE-Gene transcripts / TE-G transcripts).
3. Classify the ones contributing to alternative isoforms of genes (Alternative TE-Gene isoforms / ATE-G isoforms).

ParasiTE detects candidates for TE-AS and TE-ATP events as illustrated below using CATANA predictions:

Main steps of ParasiTE

ParasiTE is composed of five main steps:

1. Remove transcripts of gene-like TEs (transcripts of active TEs which are not involved in TE-gene transcripts)
2. Discrimination of intragenic and intergenic TEs
3. Discrimination of intronic and exonic TEs
4. Detection of TE-gene (TE-G) transcripts events
5. Detection of alternative TE-gene (ATE-G) isoform events

Prerequisites to run ParasiTE in Linux

An installation time of around 40 min if you need to install all dependencies

0. Download ParasiTE:

git clone https://github.com/JBerthelier/ParasiTE.git

1. R (versions 3.6.0 or 3.6.1) https://cran.r-project.org/src/base/R-3/ or you can use conda to install R 3.6.1

conda create -n YourEnvironment -c conda-forge r-base=3.6.1

3. R libraries:

• optparse install.packages("optparse")
• stringr install.packages("stringr")
• data.table install.packages("data.table")
• dplyr install.packages("dplyr")
• splitstackshape install.packages("splitstackshape")
• tidyr install.packages("tidyr")

3. bedtools (versions 2.27.1 and 2.29.2 were tested) you can install it locally or with conda conda install bedtools

4. BEDOPS (version 2.4.36 was tested) you can install locally or with conda conda install -c bioconda bedops

Input data

Three inputs are required and one is optional

1. TE annotation in .gff/gtf
2. gene annotation in .gff/.gtf
3. de novo transcriptome annotation in .gtf file generated by Stringtie2 (version 2.1.4 was used) (https://ccb.jhu.edu/software/stringtie/) or a gff/gtf file that has the same structure as files generated by Stringtie2 (see below Table 1).
4. [OPTIONAL] a gene-like transcript of TE annotation (transcripts of active TEs) such as the one of Panda et al. 2020 for A. thaliana

Outputs

Results are in "ParasiTE_output" directory:

• Annotation of intergenic and intragenic TEs.
• Among Intragenic TEs, the annotation of intragenic (intronic and exonic) TEs.
• List of TE-genes candidates
• List of ATE-G isoforms candidates

Run Parasite demo data to test your installation

The expected run time for demo data is 2 min (it was tested on a Linux Machine Ubuntu with 8Gb of memory)

1. extract the folder cd /ParasiTE/

tar -xvf Demo_data_Araport11.tar.gz

2. run the command

Rscript /Fullpathway/ParasiTE/ParasiTE_v1/ParasiTE.R -T /Fullpathway/ParasiTE/Demo_data_Araport11/TEs_urgi_tair10.min200.gff3 -R /Fullpathway/ParasiTE/Demo_data_Araport11/Athaliana_447_Araport11.transcript_exons.for_ParasiTE_SC.gtf -G /Fullpathway/ParasiTE/Demo_data_Araport11/Athaliana_447_Araport11.gene.gff3 -L /Fullpathway/ParasiTE/Demo_data_Araport11/TAIR10-Panda_cat_TE_gene-like.gff3 -P SC

/Fullpathway/ has to be replaced by your data pathway

If the script finished without errors and you get files in five output directories (STEP1 to STEP5) in ParasiTE_output/Results your installation is well done.

Run your data with ParasiTE (please read until the end)

The basic command is:

Rscript /Fullpathway/ParasiTE/ParasiTE_v1/ParasiTE.R -T /Fullpathway/TE_annotation.gff3 -G /Fullpathway/gene_annotation.gtf -R /Fullpathway/transcripts_annotation.gtf -L /Fullpathway/gene-like_TE_annotation.gff3 -P {mode}

ParasiTE was built to work with Stringtie2 de novo transcriptome (but can use custom transcriptomes see how to use it below).

We choose Stringtie2, because it allows to identify chimeric transcripts such as ATE-G. Moreover. Stringtie2 supports short reads (eg. Illumina) or long reads (eg. PacBio or Oxford Nanopore).

For the -P {mode}

1. Transcriptomes obtained with Stringtie2 from long reads alignment

• If the transcriptome was obtained with Stringtie2 with the -L mode you must use -P SL

• If the transcriptome was obtained with Stringtie2 with the -R mode you must use -P SR

2. Transcriptome annotation obtained with Stringtie2 from short reads alignment you must use -P SL

3. Transcriptome obtained by stringtie2 Merge option or custom transcriptome following the same format as Stringtie2 you must use -P SA

4. Custom transcriptome following the structure displayed below (such as "/Demo_data/Athaliana_447_Araport11.transcript_exons.for_ParasiTE.gtf" ) you must use -P SC

Input data structure

For gene annotation, transcriptome annotation, TE annotation and gene-like TE annotation, the seqname must be numeric (no "Chr1" but 1, no "Mit" but a number) (see Table 1)

Transcriptome annotation

ParasiTE was developed to work with Stringtie2 structure transcriptome (see Table 1) However, for some transcriptome annotations, the exon numbering differs from Stringtie2.

For any custom transcriptome (SC or SA) the attribute must be as below:

Table 1:

seqname	source	feature	start	end	score	strand	frame	attribute
1	Ref	transcript	6788	9130	1000	+	.	gene_id "ID.1"; transcript_id "ID.1.1";
1	Ref	exon	6788	7069	1000	.	.	gene_id "ID.1"; transcript_id "ID.1.1"; exon_number_id "1";
1	Ref	exon	8571	9130	1000	.	.	gene_id "ID.1"; transcript_id "ID.1.1"; exon_number_id "2";
1	Ref	transcript	3631	5899	1000	+	.	gene_id "ID.1"; transcript_id "ID.1.2";
1	Ref	exon	6788	7069	1000	.	.	gene_id "ID.1"; transcript_id "ID.1.2"; exon_number_id "1";
1	Ref	exon	7157	7450	1000	.	.	gene_id "ID.1"; transcript_id "ID.1.2"; exon_number_id "2";
1	Ref	exon	8571	8737	1000	.	.	gene_id "ID.1"; transcript_id "ID.1.2"; exon_number_id "3";

etc...

Be careful

• "seqname" must be a number shown in the table (no characters allowed, in example "1" corresponds to "chromosome 1").

-"Attribute" must follow the correct format as displayed.

-for transcript: gene_id "ID.1"; transcript_id "ID.1.1"; ("ÏD" can be any other word, such as GENE.1, transcript_id "GENE.1.1")

-for exon: gene_id "ID.1"; transcript_id "ID.1.1"; exon_number_id "1";

Otherwise, ParasiTE will not work properly

TE annotation

We used the following structure for TE annotation, you need to provide a "ID=theTEid" in the attribute (see example in Table 2)

Table 2:

seqname	source	feature	start	end	score	strand	frame	attribute
1	Ref	TE	17024	18924	.	.	.	ID=AT1TE00025;Number=594947;super_family=DHX;family=ATREP3
1	Ref	TE	18331	18642	.	.	.	ID=AT1TE00030;Number=597081;super_family=DTA;family=ATHATN7
1	Ref	TE	55676	56576	.	.	.	ID=AT1TE00150;Number=592885;super_family=DTA;family=SIMPLEHAT1

etc...

Detailled outputs

In /ParasiTE_output/Results/STEP4_TE-G_candidates/

• List_TE-G_exonic_level.tab ## List of Genes with exonic region overlaping with a TE annotation.
• List_TE-G.tab ## List of exons region that overlaping with a TE annotation.

In /ParasiTE_output/Results/STEP5_altTE-G_candidates/

• List_altTE-G.tab ## List of Genes that are involved in ATE-G isoforms.
• List_altTE-G_exonic_level.tab ## List of exons that are involved in ATE-G isoforms.

In "List_altTE-G.tab"

Column name	description
TE_gene	The id of the TE-gene event
Gene_id	The id of the gene
Total_number_transcripts	Number of isoforms transcribed by the gene
Total_transcripts_with_TE	Number of isoforms transcribed by the gene that are overlapped by the TE
total_exon_number	Number of exons
Freq_TE_isoform	Frequence of isoforms overlapped by the TEs for the gene
TE_id	The id of the TE
TE_chromosome	The Chromosome location of the TE annotation
TE_start	The start location of the TE annotation
TE_end	The end location of the TE annotation
TE_localisation	intragenic or intergenic
method	ParasiTE method used to detect the exonic TE (M1 and/or M2 and/or M3)
Alternative_splicing	The predicted AS event caused by the TE
Alternative_transcription	The predicted ATP event caused by the TE
first	Count of TE overlapping with exons of gene transcripts at the first exon (5'->3')
middle	Count of TE overlapping with exons of gene transcripts at middle exon (5'->3')
last	Count of TE overlapping with exons of gene transcripts at the last exon (5'->3')
single	Count of TE overlapping with single-exon of gene transcripts (5'->3')

Parasite help

Rscript /Fullpathway/ParasiTE.R -h

Citation

Please cite our work:

Berthelier, J., Furci, L., Asai, S. et al. Long-read direct RNA sequencing reveals epigenetic regulation of chimeric gene-transposon transcripts in Arabidopsis thaliana. Nature Communications 14, 3248 (2023). https://doi.org/10.1038/s41467-023-38954-z