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| NanoSim 1.0.0 | |
| ------------------------------------------------------------------------------------ | |
| NanoSim is a fast and scalable read simulator that captures the technology-specific | |
| features of ONT data, and allows for adjustments upon improvement of nanopore | |
| sequencing technology. | |
| ------------------------------------------------------------------------------------ | |
| ------------------------------------------------------------------------------------ | |
| Dependencies: | |
| LAST (Tested with version 581) | |
| R (Tested with version 3.2.3) | |
| Python (2.6 or above) | |
| Numpy (Tested with version 1.10.1 or above) | |
| ------------------------------------------------------------------------------------ | |
| Usage | |
| NanoSim is implemented using R for error model fitting and Python for read length | |
| analysis and simulation. The first step of NanoSim is read characterization, which | |
| provides a comprehensive alignment-based analysis, and generates a set of read | |
| profiles serving as the input to the next step, the simulation stage. The simulation | |
| tool uses the model built in the previous step to produce in silico reads for a | |
| given reference genome. It also outputs a list of introduced errors, consisting of | |
| the position on each read, error type and reference bases. | |
| 1. Characterization stage | |
| Characterization stage takes a reference and a training read set in FASTA format as | |
| input. User can also provide their own alignment file in MAF format. | |
| Usage: | |
| ./read_analysis.py <options> | |
| [options]: | |
| -h : print usage message | |
| -i : training ONT real reads, must be fasta files | |
| -r : reference genome of the training reads | |
| -m : User can provide their own alignment file, with maf extension. Optional | |
| -o : The prefix of output file, default = 'training' | |
| 2. Simulation stage | |
| Simulation stage takes reference genome and read profiles as input and outputs | |
| simulated reads in FASTA fomat. | |
| Usage: | |
| ./simulator.py [command] <options> | |
| [command]: | |
| circular | linear | |
| # Do not choose 'circular' when there is more than one sequence in the reference | |
| <options>: | |
| -h : print usage message | |
| -r : reference genome in fasta file, specify path and file name. Required | |
| -c : the prefix of training set profiles, same as the output prefix in | |
| read_analysis.py, default = training | |
| -o : The prefix of output file, default = 'simulated' | |
| -n : Number of generated reads, default = 20,000 reads | |
| --perfect: Output perfect reads, no mutations. Optional | |
| --KmerBias: prohibits homopolymers with length >= 6 bases in output reads. Optional | |
| For example: | |
| 1 If you want to simulate E. coli genome, then circular command must be chosen | |
| because it's a circular genome | |
| ./simulator.py circular -r Ecoli_ref.fasta -c ecoli | |
| 2 If you want to simulate only perfect reads, i.e. no snps, or indels, just simulate | |
| the read length distribution | |
| ./simulator.py circular -r Ecoli_ref.fasta -c ecoli --perfect | |
| 3 If you want to simulate S. cerevisiae genome with kmer bias, then linear command | |
| must be chosen because it's a linear genome | |
| ./simulator.py linear -r yeast_ref.fasta -c yeast --KmerBias | |
| See more detailed example in example.sh | |
| ------------------------------------------------------------------------------------ | |
| Explaination of output files | |
| 1. Characterization stage | |
| training_aligned_length_ecdf: Length distribution of aligned regions on aligned | |
| reads | |
| training_aligned_reads_ecdf: Length distribution of aligned reads | |
| training_align_ratio: Empirical distribution of align ratio of each read | |
| training_besthit.maf: The best alignment of each read based on length | |
| training_match.hist/training_mis.hist/training_del.hist/training_ins.hist: | |
| Histogram of match, mismatch, and indels | |
| training_first_match.hist: Histogram of the first match length of each alignment | |
| training_error_markov_model: Markov model of error types | |
| training_ht_ratio: Empirical distribution of the head region vs total unaligned | |
| region | |
| training.maf: The output of LAST, alignment file in MAF format | |
| training_match_markov_model: Markov model of the length of matches (stretches | |
| of correct base calls) | |
| training_model_profile: Fitted model for errors | |
| training_processed.maf: A re-formatted MAF file for user-provided alignment file | |
| training_unaligned_length_ecdf: Length distribution of unaligned reads | |
| 2. Simulation stage | |
| simulated.log: Log file for simulation process | |
| simulated_reads.fasta: FASTA file of simulated reads. Each reads has "unaligned", | |
| "aligned", or "perfect" in the header determining their error rate. | |
| "unaligned" means that the reads have an error rate over 90% and cannot be | |
| aligned. "aligned" reads have the same error rate as training reads. "perfect" | |
| reads have no errors. | |
| To explain the information in the header, we have two examples: | |
| a. >ref|NC-001137|-[chromosome=V]_468529_unaligned_0_F_0_3236_0 | |
| All information before the first _ are chromosome information. 468529 is the start | |
| position and unaligned suggesting it should be unaligned to the reference. The | |
| first 0 is the sequence index. F represents a forward strand. 0_3236_0 means that | |
| sequence length extracted from the reference is 3236 bases. | |
| b. >ref|NC-001143|-[chromosome=XI]_115406_aligned_16565_R_92_12710_2 | |
| This is an aligned read coming from chromosome XI at position 115406. 16565 is | |
| the sequence index. R represents a reverse complement strand. 92_12710_2 | |
| means that this read has 92-base head region (cannot be aligned), followed by | |
| 12710 bases of middle region, and then 2-base tail region. | |
| The information in the header can help users to locate the read easily. | |
| simulated_error_profile: Contains all the information of errors introduced into | |
| each reads, including error type, position, original bases and current bases. |