PINTS is available on PyPI and bioconda, which means you can install PINTS easily with:

pip install pyPINTS

or

conda install bioconda::pypints

Alternatively, you can clone this repo to a local directory, then run the following command in that directory:

python setup.py install

PINTS can call peaks from either bigWig or BAM files. If you have signals for the forward and reverse strands in two separate bigWig files (path_to_pl.bw and path_to_mn.bw), you can use command like the following to get the peaks:

pints_caller --save-to output_dir \
  --file-prefix output_prefix \
  --bw-pl path_to_pl.bw \
  --bw-mn path_to_mn.bw \
  --thread 16

To call peaks from BAM files: you'll need to provide PINTS a path to the BAM file and what kind of experiment it was from. If it's from a standard protocol, like PROcap, then you can set --exp-type PROcap. Other supported experiments including GROcap/ CoPRO/ csRNAseq/ NETCAGE/ CAGE/ RAMPAGE/ STRIPEseq. For a comprehensive list of directly supported assays, please run

pints_caller --help

If the data was generated by other methods, you need to tell PINTS where it can find ends of RNAs you are interested in. For example, --exp-type R_5 tells the tool that:

1. this alignment is from a single-end library;
2. the tool should look at 5' of reads. Other supported values are R_3, R1_5, R1_3, R2_5, R2_3.

If reads represent the reverse complement of original RNAs, like PROseq, then you need to use --reverse-complement (not necessary for standard protocols).

One example for calling peaks from BAM file:

pints_caller --bam-file input.bam \
  --save-to output_dir \
  --file-prefix output_prefix \
  --thread 16 \
  --exp-type PROcap

We have prepared several case studies demonstrating steps from processing the raw fastq files to calling peaks/TREs for your reference.

• prefix+_{SID}_divergent_peaks.bed: Divergent TREs;
• prefix+_{SID}_bidirectional_peaks.bed: Bidirectional TREs (divergent + convergent);
• prefix+_{SID}_unidirectional_peaks.bed: Unidirectional TREs, maybe lncRNAs transcribed from enhancers (e-lncRNAs) as suggested here.

{SID} will be replaced with the number of samples that peaks are called from, if you only provide PINTS with one sample, then {SID} will be replaced with 1, if you try to use PINTS with three replicates (--bam-file A.bam B.bam C.bam), then {SID} for peaks identified from A.bam will be replaced with 1.

For divergent or bidirectional TREs, there will be 6 columns in the outputs:

1. Chromosome
2. Start site: 0-based
3. End site: 0-based
4. Confidence about the peak pair. Can be:
   • Stringent(qval), which means the two peaks on both forward and reverse strands are significant based on their q-values;
   • Stringent(pval), which means one peak is significant according to q-value while the other one is significant according to p-value;
   • Relaxed, which means only one peak is significant in the pair.
   • A combination of the three types above, because of overlap for nearby elements.
   • If epigenomic annotation is enabled by --epig-annotation <biosample>, then peaks that are less significant (--relaxed-fdr-target, default is 2*fdr_target), but overlap with epigenomic annotations from PINTS web server, will be listed with the confidence level: Marginal.
5. Major TSSs on the forward strand, if there are multiple major TSSs, they will be separated by comma ,
6. Major TSSs on the reverse strand, if there are multiple major TSSs, they will be separated by comma ,

For unidirectional TREs, there will be 9 columns in the output:

1. Chromosome
2. Start
3. End
4. Peak ID
5. Q-value
6. Strand
7. Read counts
8. Position of the summit TSS
9. Height of the summit

For all three types of TREs, if a valid biosample name for --epig-annotation is provided, then an additional column with epigenomic annotation for each TRE will show up in the final output.

• If you want to use BAM files as inputs:
  • --bam-file: input bam file(s);
  • --exp-type: Type of experiment. If the experiment is not listed as a choice, or you know the position of RNA ends on the reads and you want to override the defaults, you can specify:
    • R_5 (5' of the read for single-end lib),
    • R_3 (3' of the read for single-end lib),
    • R1_5 (5' of the read1 for paired-end lib),
    • R1_3 (3' of the read1 for paired-end lib),
    • R2_5 (5' of the read2 for paired-end lib),
    • or R2_3 (3' of the read2 for paired-end lib)
  • --reverse-complement: Set this switch if 1) exp-type is Rx_x and 2) reads in this library represent the reverse complement of RNAs, like PROseq;
  • --ct-bam: Bam file for input/control (optional);
• If you want to use bigwig files as inputs:
  • --bw-pl: Bigwig for signals on the forward strand;
  • --bw-mn: Bigwig for signals on the reverse strand;
  • --ct-bw-pl: Bigwig for input/control signals on the forward strand (optional);
  • --ct-bw-mn: Bigwig for input/control signals on the reverse strand (optional);
• --save-to: save peaks to this path (a folder), by default, current folder
• --file-prefix: prefix to all outputs

• --dont-merge-reps: Starting with PINTS 1.2.x, the software automatically merges multiple replicates for a joint peak calling process. To call peaks individually for each sample, as in previous versions, use this option.
• --epig-annotation <biosample>: Use this option together with the name of the biosample that the library was derived from, for example K562; then epigenomic annotations will be downloaded from the PINTS web server and used for annotating and augmenting TREs identified by PINTS (for hg38 only);
• --relaxed-fdr-target <relaxed fdr>: In the presence of --epig-annotation, peaks that do not pass the original FDR cutoff but pass this relaxed cutoff and have support from DNase-seq and H3K27ac ChIP-seq will also be included in final outputs. By default, 2*fdr;
• --mapq-threshold <min mapq>: Minimum mapping quality, by default: 30 or None;
• --close-threshold <close distance>: Distance threshold for two peaks (on opposite strands) to be merged, by default: 300;
• --fdr-target <fdr>: FDR target for multiple testing, by default: 0.1;
• --chromosome-start-with <chromosome prefix>: Only keep reads mapped to chromosomes with this prefix. By default, all reads will be analyzed;
• --thread <n thread>: Max number of threads the tool can create;
• --borrow-info-reps: Borrow information from reps to refine calling of divergent elements;
• --sensitive: Call peaks in a more sensitive mode (LRT+FC).

More parameters can be seen by running pints_caller -h.

In this section, we try to identify differentially expressed TREs (promoters and enhancers) from two conditions.

First, call peaks for each condition with pints_caller:

# control samples
pints_caller --bw-pl DMSO_r1_pl.bw DMSO_r2_pl.bw \ 
  --bw-mn DMSO_r1_mn.bw DMSO_r2_mn.bw \
  --thread 16 --file-prefix DMSO
# and treatment samples
pints_caller --bw-pl E2_r1_pl.bw E2_r2_pl.bw \
  --bw-mn E2_r1_mn.bw E2_r2_mn.bw \
  --thread 16 --file-prefix E2

Second, build the counts table with pints_counter:

pints_counter -b DMSO_1_bidirectional_peaks.bed E2_1_bidirectional_peaks.bed \ 
  -u DMSO_1_unidirectional_peaks.bed E2_1_unidirectional_peaks.bed \
  -p DMSO_r1_pl.bw DMSO_r2_pl.bw E2_r1_pl.bw E2_r2_pl.bw \
  -m DMSO_r1_mn.bw DMSO_r2_mn.bw E2_r1_mn.bw E2_r2_mn.bw \
  -c DMSO DMSO E2 E2 \
  -r 1 2 1 2 \
  -s counts.csv

The counts table look like the following:

,DMSO_1,DMSO_2,E2_1,E2_2
chr1:10609-10620,17,22,44,43
chr1:629905-629938,169,13,224,82
chr1:633956-634096,218,12,271,102
chr1:778554-778929,1180,195,1327,495
chr1:779719-779721,0,0,12,2
chr1:779846-780119,6,0,30,6
chr1:827199-827316,48,22,101,46
chr1:827326-827736,634,88,752,318
chr1:827742-827773,19,0,32,8

Third, feed DESeq2/edgeR with the counts table for differential expression analysis

• pints_visualizer: Generate bigwig files for the inputs.
• pints_counter: Generate count matrix for downstream usages (e.g. differential expression analysis).
• pints_normalizer: Normalize inputs.
• pints_boundary_extender: Extend peaks from summits.

You can use tool_name --help to see parameters for each tool.

• Citation: If you use PINTS in your work, please cite: https://www.nature.com/articles/s41587-022-01211-7.
• Support: Please submit an issue with any questions or if you experience any issues/bugs.