ART_454 Version 2.6.0 README, last updated on 03/18/2015, Weichun Huang at whduke@gmail.com DESCRIPTION ART_454 is a simulation program to generate sequence read data of Roche 454 Pyrosequencing sequencers. ART generates reads according to the empirical read quality profile and the calibrated error profile of uncall/overcall homopolymers from real 454 read data. ART has been using for testing or benchmarking a variety of method or tools for next-generation sequencing data analysis, including read alignment, de novo assembly, detection of SNP, CNV, or other structure variation. art_454 can generate both single-end and paired-end of 454 sequencing platform. Besides for regular genome DNA and cDNA sequencing simulation, art_454 also supports amplicon sequencing. The reference sequences can be either DNA or RNA. ART outputs include FASTQ read, ALN alignment, STAT read coverage, and optional SAM alignment files. ALN files can also be converted to UCSC BED files by using aln2bed.pl program included. art_454 comes with art_profiler_454, the tool for generating a art_454 read profile from 454 sequencing data in the fastq format. The tool in the folder ART_profiler_454. Please see the README under in the folder for details EXAMPLES In the "examples" subdirectory, the shell script "run_test_examples_454.sh" gives two examples of using art_454 for read simulation. To test these two examples, just run the script "run_test_examples_454.sh" USAGES SINGLE-END SIMULATION art_454 [-s] [-a ] [-t] [-r rand_seed] [ -p read_profile ] [ -c num_flow_cycles ] PAIRED-END SIMULATION art_454 [-s] [-a ] [-t] [-r rand_seed] [ -p read_profile ] [ -c num_flow_cycles ] AMPLICON SEQUENCING SIMULATION art_454 [-s] [-a ] [-t] [-r rand_seed] [ -p read_profile ] [ -c num_flow_cycles ] <-A|-B> <#_READS/#_READ_PAIRS_PER_AMPLICON> ===== PARAMETERS ===== INPUT_SEQ_FILE - the filename of DNA/RNA reference sequences in FASTA format OUTPUT_FILE_PREFIX - the prefix or directory of output read data file (*.fq) and read alignment file (*.aln) FOLD_COVERAGE - the fold of read coverage over the reference sequences MEAN_FRAG_LEN - the average DNA fragment size for paired-end read simulation STD_DEV - the standard deviation of the DNA fragment size for paired-end read simulation #READS_PER_AMPLICON - number of reads per amplicon (for 5'end amplicon sequencing) #READ_PAIRS_PER_AMPLICON - number of read pairs per amplicon (for two-end amplicon sequencing) ===== OPTIONAL PARAMETERS ===== -A indicate to perform single-end amplicon sequencing simulation -B indicate to perform paired-end amplicon sequencing simulation -M indicate to use CIGAR 'M' instead of '=/X' for alignment match/mismatch -a indicate to output the ALN alignment file -s indicate to output the SAM alignment file -d print out warning messages for debugging -t indicate to simulate reads from the built-in GS FLX Titanium profile [default: GS FLX profile] -r specify a fixed random seed for the simulation (to generate two identical datasets from two different runs) -c specify the number of flow cycles by the sequencer [ default: 100 for GS-FLX, and 200 for GS-FLX Titanium ] -p specify user's own read profile for simulation NOTE: the name of a read profile is the directory containing read profile data files. please read the REAME file about the format of 454 read profile data files and. and the default filenames of these data files. ===== EXAMPLES ===== 1) singl-end simulation with 20X coverage art_454 -s seq_reference.fa ./outdir/single_dat 20 2) paired-end simulation with the mean fragment size 500 and STD 20 using GS FLX Titanium platform art_454 -s -t seq_reference.fa ./outdir/paired_dat 10 1500 20 3) paired-end simulation with a fixed random seed art_454 -s -r 777 seq_reference.fa ./outdir/paired_fxSeed 10 2500 50 4) single-end amplicon sequencing with 10 reads per amplicon art_454 -A -s amplicon_ref.fa ./outdir/amp_single 10 5) paired-end amplicon sequencing with 10 read pairs per amplicon art_454 -B -s amplicon_ref.fa ./outdir/amp_paired 10 READ PROFILE FILES CREATE A NEW PROFILE Please use the tool art_profiler_454 included in the distribution to generate new art_454 read profiles from new 454 sequencing data in the fastq format. The tool in the folder ART_profiler_454. Please see the README under in the folder for usage. 454 READ PROFILE The name of a read profile is the filename of directory containing read profile data files. a read profile should consist of the three required profile files, and one optional profile file. The three REQUIRED profiles are: qual_1st_profile, qual_mc_profile, length_dist. 1) qual_1st_profile - the empirical homopolymer-length dependent quality distribution of the 1st base of DNA homopolymers 2) qual_mc_profile - 1st order Markov Chain-based empirical quality distribution of the remaining bases of DNA homopolymers 3) length_dist - the empirical distribution of 454 read length The OPTIONAL profile is indel_erro_profile. When the profile is not given, the built-in indel_erro_profile will be used. indel_erro_profile - undercall or overcall error profiles of DNA homopolymers FILE FORMATS all read profile files are tab-delimited text files. For detailed formats, please see the real examples the under the folder "454_profiles/GS_FLX_profile" or "454_profiles/GS_FLX_Titanium_profile" for file formats of these four files. CREATION OF YOUR OWN 454 READ PROFILE Users can use the program "454_readprofile_art" that comes with the software package to generate 454 read profile from 454 sequencing data in the fastq format. USAGE: ./454_readprofile_art input_fastq_files_dir output_profile_dir [fastq_filename_extension] PARAMETERS 1) input_fastq_files_dir - directory containing all 454 read data files in the fastq format 2) output_profile_dir - directory to put read profile files generated by the program EXAMPLES: perl 454_readprofile_art 454_dat_dir 454_profile_dir perl 454_readprofile_art 454_dat_dir 454_profile_dir fastq NOTES: 1)the default filename extension for fastq files is fq 2)the program can read gzipped fastq data files with filename extension fq.gz OUTPUT FILES *.fq - FASTQ read data files. For paired-end read simulation, *1.fq contains data of the first reads, and *2.fq for the second reads. *.aln - read alignment files. For paired-end read simulation, *1.aln has read alignments for the first reads and *2.aln for the second reads. *.sam - SAM alignment file. *.stat - read coverage files with the following three tab-delimited fields per lines: (1)reference position (2)number of reads starting at the position (3)number of reads covering the position FASTQ FILE format A FASTQ file contains both sequence bases and quality scores of sequencing reads and is in the following format: @read_id sequence_read + base_quality_scores A base quality score is coded by the ASCII code of a single character, where the quality score is equal to ASCII code of the character minus 33. Example: @refid-4028550-1 caacgccactcagcaatgatcggtttattcacgat... + ????????????7???????=&?<ref_seq_id read_id aln_start_pos ref_seq_strand ref_seq_aligned read_seq_aligned aln_start_pos is the alignment start position of reference sequence. aln_start_pos is always relative to the strand of reference sequence. That is, aln_start_pos 10 in the plus (+) strand is different from aln_start_pos 10 in the minus (‐) stand. ref_seq_aligned is the aligned region of reference sequence, which can be from plus strand or minus strand of the reference sequence. read_seq_aligned is the aligned sequence read, which always in the same orientation of the same read in the corresponding fastq file. SAM format SAM is a standard format for next-gen sequencing read alignments. The details of the format and examples are available at the links below: 1) http://samtools.sourceforge.net/SAM1.pdf 2) http://genome.sph.umich.edu/wiki/SAM BED format See the format at UCSC http://genome.ucsc.edu/FAQ/FAQformat.html#format1 NOTE: both ALN and BED format files use 0-based coordinate system while SAM format uses 1-based coordinate system.