EAHelitron  

Easy-to-Annotate Helitrons Unix-like Command-Line           

EAHelitron is written in Perl. Used the Helitron conservative structure traits: 5’ terminal with TC, 3’ terminal with CTAGt, and before CTAG 2-10 nt has a GC-rich hairpin loop. We used the Perl regular expression(RE) engine and its Embedded-Code Construct to find all the matches and then printed and converted them to a GFF3 format file. Using the above GFF3 file, we can visualize these Helitrons in genome visualization tools such as IGV, Gbrowse, and Jbrowse, and easily characterize the captured genes.

EAHelitron is a Unix-like program compatible with all Perl 5.10+ supported machines, allowing command-line usage and piping. Tests have been passed on Linux, Mac OS, and Windows.

New EAHelitronMFE using RNAfold to calculate the minimus free energy (MFE) to filter the haipins structure.

Getting Started

Prerequisites

Ensure that Perl is installed on your system. Verify by typing the following command in your terminal:

perl -v

If Perl version information is displayed, you can proceed to download the EAHelitron files from https://github.com/dontkme/EAHelitron/archive/master.zip

Installing

Simply unzip the downloaded zip file:

unzip EAHelitron-master.zip

Navigate to the extracted folder and run EAHelitron:

cd EAHelitron-master
perl EAHelitron -h

If the screen displays help and version information. It works.

Running

Basic mode：

Example: Predict Helitrons and search for their 5'TC terminals in the upstream 20,000bp range.

perl EAHelitron –o testEAHout –u 20000 teat.fas

or

./EAHelitron –o testEAHout –u 20000 teat.fas

Options:

     [-o string|outprefix Default: EAHeli_out]
     [-u int|upstream length Default: 3000]
     [-d int|downstream length Default: 500]

     Advanced options:
     [-T string|TC pattern. User's 5'TC pattern]
     [-H string|Hairpin pattern. User's Hairpin left pattern]
     [-E float|minimum free energy (MFE) threshold, default: -5]
     [-r int[0-5]|CTRRt 3' terminal fuzzy level;
             0: CTAGT
             1: CT[AG]GT
             2: CTA[AG]T
             3: CT[AG]{2}T
             4: CT[AG]{2}.{1}
             5: CTAG.{1}
             Default: 0]

We also provide EAHelitron_P, a multi-threaded version that can speed up running in a large genome.

(Need Perl Parallel::ForkManager. You could install it by command: cpan Parallel::ForkManager )

perl EAHelitron_P –p 8 –o testEAHout –u 20000 teat.fas

-p: How many threads to use. It is recommended not to exceed the number of sequences contained in the fasta file you input.

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Advanced options:

Warning: Advanced options may significantly increase the false positive rate, only for exploring!!!

-r: CTRRt 3' terminal fuzzy level:

6 fuzzy levels of CTRRt terminal [0-5]

perl EAHelitron_P –p 8 -r 3 –o testEAHout_r3 teat.fas

Users can enter their own patterns (Perl RE) to predict Helitrons.

-H: Use Hairpin left sequence RE pattern:

Example:

1. Only use a GC as hairpin left sequence pattern:

perl EAHelitron_P –p 8 -H "GC" –o testEAHout_H_GC teat.fas

2. or use 1 GC(G or C) 1 AT(A or T) and 5 GC or(|) 6 GC as haripin left sequence pattern:

perl EAHeliton_P –p 8 -H "[GC]{1}[AT]{1}[GC]{5}|[GC]{6}" –o testEAHout_H_GC teat.fas

-T: Use 5' TC sequence RE pattern:

Example:

1. Only use 'TC' as 5' TC sequence RE pattern:

perl EAHelitron_P –p 8 -T "TC" –o testEAHout_T_TC teat.fas

2. Use TC or(|) TCT.TACTA.T as 5' TC sequence RE pattern (The dot '.' to indicate any character, we can use [ATCGN] instead of '.', if you like):

perl EAHeliton_P –p 8 -T "TC|TCT.TACTA.T" –o testEAHout_T teat.fas

We can also use them in combination:

perl EAHelitron_P –p 8 -T "TC" -H "GC" –o testEAHout_T_H teat.fas

Or more complex:

perl EAHeliton_P –p 8 -H "[GC]{1}[AT]{1}[GC]{5}|[GC]{6}" -T "TC|TCT.TACTA.T" –o testEAHout_T_H teat.fas

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-E: minimum free energy (MFE) threshold, default: -5 (Only avaliable for EAHelitronMFE).

EAHlitronMFE version could using RNAfold to filter the haipin minimum free energy (MFE).

Please make sure RNAfold could be running in your environment. If the terminal shows the RNAfold version information after you typing this command, you could run the EAHlitronMFE.

RNAfold -V

Example:

Using two G or C as hairpin left sequence pattern and haipin need a MFE less than -5.5:

perl EAHelitronMFE -T "[GC]{2}" -E -5.5 –o testEAHout_GC_E_5 teat.fas

Outputs

The outputs are named like EAHout.3.txt EAHout.5.txt EAHout.5.fa EAHout.gff3 EAout.u20000.fas. (The prefix 'EAHout' can be set with the -o option, 20000 is the value of your -u option)

*.3.txt: All 3’ terminal sequences with a 10nt left flank and a 4nt right flank in fasta format. All sequences are named by their local chromosome name, an 'H' means Helitron, '.3' suffix to mark they are 3’ terminals. The minus strain terminals have a 'tr' prefix. (e.g. Chr1H10.3, trChr5H40.3).

*.5.txt: All 5’terminal sequences were matched in the 3’ terminal’s upstream sequences, with a 5 nt left flank and a 20 nt right flank. The names of sequences have .5.1 suffix to mark they are 5’ terminal and the match orders numbers. (e.g. Chr1H10.5.1,trChr5H40.5.2)

*.5.fa: Possible full-length Helitron sequences which start with 5’ terminal and end with 3’ terminal. (Only for Helitrons)

.u.fas: All 3’ terminal upstream sequences.

.d.fas: All 3’ terminal downstream sequences.

*.gff3: Helitron location information in GFF3 format.

*.bed: Helitron 3'-ends location in bed format.

*.len.txt: Summary of genome sequences length, Helitron counts, and Helitron Densities

History

(EAHelitronMFE) v1.6000 2023/10/05 New strategy, using RNAfold to calculate the minimus free energy (MFE) to filter the haipins. Test in Arabidopsis thaliana (TAIR10) genome, -T [GC]{2} -E -5 options get 7532 candidate Helitrons.

(EAHelitron) v1.5400 2022/05/27 New default hairpin-left-sequence pattern (allow 2 [AT] in 5 [GC]). The default results for A. thaliana increased from 665 to 708, and the false positive rate increased slightly from 5.91% to 6.47%.

(EAHelitron) v1.5300 2021/06/25 Add a feature. User TC pattern and hairpin left sequence pattern options.

(EAHelitron) v1.5200 2020/09/25 Use a new regular expression, which is based on BioPerl, to get chromosome names, in order to adapt to more cases. Thanks to Darcy Jones's advice.

(EAHelitron) v1.5100 2019/06/10 Add a feature. Output a BED of 3’-ends. (*.bed)

(EAHelitron) v1.5000 2019/03/08 Add a feature. Output a summary of genome sequences length, Helitron counts and Helitron Densities. (*.len.txt)

(EAHelitron) v1.4000 2018/08/31 Add CTAGT fuzzy level [0-5] option.

(EAHelitron) v1.3100 2017/09/19 Add snp switch.

(EAHelitron)v1.3000 2017/08/29 Add a feature for downstream sequences.

...

(EAHelitron) v1.0000 2016/09/22 first version upload to GitHub.

Citation

If you've found EAHelitron useful in your work, please cite our paper:

Hu, K. et al. Helitron distribution in Brassicaceae and whole genome Helitron density as a character for distinguishing plant species. BMC bioinformatics 20, 1-20, doi:ARTN 354 10.1186/s12859-019-2945-8 (2019).

Author

Hu Kaining - Initial work - dontkme

License

This project is licensed under the MIT License - see the LICENSE file for details