Click here to download all the scripts and example data: PhasiRNAnalyzer.zip

- Format conversion and data normalization: ./phasiRNAnalyzer format

- Filter reads according to the length and count: ./phasiRNAnalyzer filter

- Phased siRNA and PHAS gene prediction: ./phasiRNAnalyzer phase

- Phase-initiator prediction: ./phasiRNAnalyzer initiator

- Data Visualization: ./phasiRNAnalyzer tool

Python 2.7.15 or 2.7.X https://www.python.org/

Python packages:

    - numpy 1.16.6: http://www.numpy.org/

    - matplotlib 2.2.5: https://pypi.org/project/matplotlib/

    - biopython 1.7.6: https://biopython.org/

Perl 5.22.1 or 5.22.X https://www.perl.org/

Bowtie 1.0.0 http://bowtie-bio.sourceforge.net/index.shtml

fasta-36.3.8g https://github.com/wrpearson/fasta36/

    # GLIBC_2.14 is required for fasta-36.3.8g, check the package is installed by "strings /lib64/libc.so.6 | grep GLIBC_"

    - GLIBC_2.14 http://www.gnu.org/software/libc/

1) Install package

    unzip phasiRNAnalyzer.zip

    cd phasiRNAnalyzer

2) Data pre-processing

    Format conversion: ./phasiRNAnalyzer format -i ./data/example.txt -it g -n 1000000 -ot fn -o 1_example_norm.fa

    Filter reads: ./phasiRNAnalyzer filter -i 1_example_norm.fa -min 16 -max 26 -count 1.0 -o 2_example_norm_filter.fa

3) Build index for cDNA or genome library

    For cDNA library: ./phasiRNAnalyzer bowtie-build ./data/oryza_sativa_cdna.fa ./index/oryza_sativa_cdna_index

    For genome library: ./phasiRNAnalyzer bowtie-build ./data/oryza_sativa_genome_chr12.fa ./index/oryza_sativa_genome_chr12_index

4) Mapping of small RNA

    For cDNA library: ./phasiRNAnalyzer bowtie -t -v 0 -p 8 -m 10 -f -a ./index/oryza_sativa_cdna_index 2_example_norm_filter.fa 3_example_norm_filter.map

    For genome library: ./phasiRNAnalyzer bowtie -t -v 0 -p 8 -m 10 -f -a ./index/oryza_sativa_genome_chr12_index 2_example_norm_filter.fa 3_example_norm_filter.map

5) Phased siRNA and PHAS gene prediction

    For cDNA library: ./phasiRNAnalyzer phase -i 3_example_norm_filter.map -o 4_example_results_tmp.21.xls -all 4_example_results_total.21.xls -l 21 -p 0.001 -m 4 -s 0 -n 0 -c -a ./data/oryza_sativa_cdna_annotation.txt -fin ./data/oryza_sativa_cdna.fa -fout 4_example_PHAS_gene.fa

    For genome library: ./phasiRNAnalyzer phase -i 3_example_norm_filter.map -o 4_example_results_tmp.21.xls -all 4_example_results_total.21.xls -l 21 -p 0.001 -m 4 -s 0 -n 0 -j ./data/oryza_sativa_genome_chr12_annotation.gff3 -fin ./data/oryza_sativa_genome_chr12.fa -fout 4_example_PHAS_gene.fa

6) Get targets of phase-initiator from putative PHAS gene:

    # If the file (5_example_prediction.txt) already exists, please delete or change the name of it.

    Offline: ./phasiRNAnalyzer prediction -q ./data/oryza_sativa_miRNA.fa -b 4_example_PHAS_gene.fa -o 5_example_prediction.txt -t -T 4 -f 6

    Online: Please upload all possible phase-initiator (usually miRNA) and PHAS gene to psRNAtarget, and get putative targets of phase-initiator. The downloadable predictions are required for the next step.

7) Phase-initiator prediction

    ./phasiRNAnalyzer initiator -i 4_example_results_tmp.21.xls -j 5_example_prediction.txt -pd 105 -pl 21 -ps 1 -o 6_example_initiator.txt

8) Visualization of results

    Single run: ./phasiRNAnalyzer tool -r 2 -l 21 -q 10 -c data/oryza_sativa_cdna.fa -i 4_example_results_tmp.21.xls -j 4_example_results_total.21.xls -m 6_example_initiator.txt

    Batch run:
    for i in `awk '{print $NF}' 4_example_results_tmp.21.xls | sort | uniq`
    do
        ./phasiRNAnalyzer tool -r $i -l 21 -q 10 -c ./data/oryza_sativa_cdna.fa -i 4_example_results_tmp.21.xls -j 4_example_results_total.21.xls -m 6_example_initiator.txt
    done