- Python (>= 3.7)
- edlib (>=1.3.8)
Option 1: use git clone and run NanoPreP as a python module without installation
git clone https://github.com/Woodformation1136/NanoPreP.git
cd NanoPreP
python -m NanoPreP --help
Option 2: use pip install
pip install nanoprep-ffm
nanoprep --help
NanoPreP optimizes adapter/primer identification parameters for each input file. During the optimization process, NanoPreP search for the best combination of (1) the adapter/primer substring used for alignment, (2) sequence similarity cutoff, and (3) aligned location cutoff that achieves the highest
The
The general usage of NanoPreP to get high-quality, non-chimeric, full-length, strand-reoriented, adapter/primer-removed, polyA-removed reads:
nanoprep \
--input_fq input.fq
--beta 0.1 \
--p5_sense 5_PRIMER_SEQUENCE \
--p3_sense A{100}3_PRIMER_SEQUENCE \
--trim_adapter \
--trim_poly \
--output_full_length output.fq \
--report report.json
-
--input_fq input.fq← file contains raw sequences -
--beta 0.1← optimize adapter/primer identification parameters using$F_{0.1}$ score -
--p5_sense 5_PRIMER_SEQUENCE← 5' primer sequence in sense strand direction -
--p3_sense A{100}3_PRIMER_SEQUENCE← expected length of polyA + 3' primer sequence in sense strand direction (see section How to specify adapter/primer and polyA/T sequences) -
--output_full_length output.fq← write full-length reads tooutput.fq -
--report report.json← write details of the run toreport.json
After running this command, two output files output.fq and report.json will be written to your working directory.
The report.json records start/stop times, the parameters used, and the detail information of the input FASTQ file.
The output.fq contains full-length reads processed by NanoPreP. For each processed read, NanoPreP appends the information of the read to the ID line (the line started with @):
@read_1 strand=0.91 full_length=1 fusion=0 ploc5=0 ploc3=0 poly5=0 poly3=-20
AGAGGCTGGCGGGAACGGGC......TTTCAAAGCCAGGCGGATTC
+
+,),+'$)'%671*%('&$%......((&'(*($%$&%&$-((84*
As shown in the example above, several flags are used for the describe a read:
| flag | regex | default | explanation |
|---|---|---|---|
strand |
-?\d+.\d* | 0 | 0: unknown; > 0: sense; < 0: antisense |
full_length |
[0|1] | 0 | 0: non-full-length; 1: full-length |
fusion |
[0|1] | 0 | 0: non-chimeric/-fusion; 1: chimeric/fusion |
ploc5 |
-?\d+ | -1 | -1: unknown; 0: removed; > 0: 5' adapter/primer location |
ploc3 |
-?\d+ | -1 | -1: unknown; 0: removed; > 0: 3' adapter/primer location |
poly5 |
-?\d+ | -1 | 0: unknown; > 0: 5' polymer length; < 0: trimmed 5' polymer length |
poly3 |
-?\d+ | -1 | 0: unknown; > 0: 3' polymer length; < 0: trimmed 3' polymer length |
According to the flags, the example "read1" is a sense strand (strand=0.91), full-length (full_length=1), non-chimeric (fusion=0), adapter/primer removed (ploc5=0 ploc3=0), and polyA removed (poly3=-20) read.
Users need to provide the adapter/primer (and polyA/T) sequences to be searched for using options --p5_sense and --p3_sense.
For example, the following command means that the 5' and 3' adatper/primer sequences on the sense strand are 'CATTC' and 'GACTA', respectively.
--p5_sense CATTC --p3_sense GACTA
If users wish to detect polyA/T tails, a pattern N{M} can be used to specify the location and length of polyA/T tails. The command below tells NanoPreP that there are poly"A" tails of a maximum length of "50" bases next to the 3' adapters/primers.
--p5_sense CATTC --p3_sense A{50}GACTA
usage: nanoprep [-h] [--version] --input_fq str [--config str] [--report str] [--processes int] [--batch_size int] [--seed int] [-n int] [--beta float] [--disable_annot] [--skip_lowq float] [--skip_short int] [--p5_sense str] [--p3_sense str]
[--isl5 int int] [--isl3 int int] [--pid5 float] [--pid3 float] [--pid_body float] [--poly_w int] [--poly_k int] [--keep_adapter] [--keep_poly] [--filter_lowq float] [--filter_short int] [--orientation int] [--output_fusion str]
[--output_truncated str] [--output_full_length str] [--suffix_filtered str]
options:
-h, --help show this help message and exit
--version show program's version number and exit
--input_fq str input FASTQ
--config str use the parameters in this config file (JSON)
--report str output report file (JSON)
--processes int number of processes to use (default: 16)
--batch_size int number of records in each batch (default: 1000000)
--seed int seed for random number generator (default: 42)
-n int max number of reads to sample during optimization (default: 100000)
--beta float the beta parameter for the optimization (default: .1)
--skip_lowq float skip low-quality reads (default: 7)
--skip_short int skip too-short reads (default: 0)
--p5_sense str 5' sense adatper/primer + polyA sequences
--p3_sense str 3' sense adatper/primer + polyA sequences
--isl5 int int ideal searching location for 5' adapter/primer sequences (default: optimized)
--isl3 int int ideal searching location for 3' adapter/primer sequences (default: optimized)
--pid5 float 5' adapter/primer percent identity cutoff (default: optimized)
--pid3 float 3' adapter/primer percent identity cutoff (default: optimized)
--pid_body float adapter/primer percent identity cutoff (default: optimized)
--poly_w int window size for polyA/T identification
--poly_k int number of A/T to be expected in the window
--trim_adapter use this flag to trim adatper/primer sequences
--trim_poly use this flag to trim polyA/T sequences
--filter_lowq float filter low-quality reads after all trimming steps (default: 7)
--filter_short int filter too short reads after all trimming steps (default: 0)
--orientation int re-orient reads (0: generic , 1: sense (default), -1: antisense)
--output_fusion str output fusion/chimeric reads to this file (use '-' for stdout)
--output_truncated str
output truncated/non-full-length reads to this file (use '-' for stdout)
--output_full_length str
output full-length reads to this file (use '-' for stdout)
--suffix_filtered str
output filtered reads with the suffix