Usage: flash [OPTIONS] MATES_1.FASTQ MATES_2.FASTQ DESCRIPTION: FLASH (Fast Length Adjustment of SHort reads) is an accurate and fast tool to merge paired-end reads that were generated from DNA fragments whose lengths are shorter than twice the length of reads. Merged read pairs result in unpaired longer reads. Longer reads are generally more desired in genome assembly and genome analysis processes. FLASH cannot merge paired-end reads that do not overlap. It also cannot merge jumping read pairs that have an outward orientation (but these reads tend not to overlap anyway). FLASH also is not designed for data that has a significant portion of indel errors (such as Sanger sequencing data). MANDATORY INPUT: To run FLASH, you may provide two FASTQ files of paired-end reads where corresponding paired reads are in the same order in both files. Alternatively, you may provide one FASTQ file, which may be standard input, containing interleaved paired-end reads (see the --interleaved option). The input FASTQ files may be either plain-text or compressed with gzip. Other compression formats for the input files are not yet supported. OUTPUT: The default output of FLASH is a FASTQ file containing the extended fragments produced by combining read pairs, two FASTQ files containing read pairs that were not combined, and histogram files that show the distribution of lengths of the extended fragments. Writing the uncombined read pairs to an interleaved FASTQ file is also supported. Also, writing the extended fragments directly to standard output is supported. Plain-text and gzip output formats are natively supported; other output formats are supported indirectly via the --compress-prog option. (Note that this is all FASTQ.) OPTIONS: -m, --min-overlap=NUM The minimum required overlap length between two reads to provide a confident overlap. Default: 10bp. -M, --max-overlap=NUM Maximum overlap length expected in approximately 90% of read pairs. It is by default set to 70bp, which works well for 100bp reads generated from a 180bp library, assuming a normal distribution of fragment lengths. Overlaps longer than the maximum overlap parameter are still considered as good overlaps, but the mismatch density (explained below) is calculated over the first max_overlap bases in the overlapped region rather than the entire overlap. Default: 70bp, or calculated from the specified read length, fragment length, and fragment length standard deviation. -x, --max-mismatch-density=NUM Maximum allowed ratio between the number of mismatched base pairs and the overlap length. Two reads will not be combined with a given overlap if that overlap results in a mismatched base density higher than this value. Note: Any occurence of an 'N' in either read is ignored and not counted towards the mismatches or overlap length. Our experimental results suggest that higher values of the maximum mismatch density yield larger numbers of correctly merged read pairs but at the expense of higher numbers of incorrectly merged read pairs. Default: 0.25. -p, --phred-offset=OFFSET The smallest ASCII value of the characters used to represent quality values of bases in FASTQ files. It should be set to either 33, which corresponds to the later Illumina platforms and Sanger platforms, or 64, which corresponds to the earlier Illumina platforms. Default: 33. -r, --read-len=LEN -f, --fragment-len=LEN -s, --fragment-len-stddev=LEN Average read length, fragment length, and fragment standard deviation. These are convenience parameters only, as they are only used for calculating the maximum overlap (--max-overlap) parameter. The maximum overlap is calculated as the overlap of average-length reads from an average-size fragment plus 2.5 times the fragment length standard deviation. The default values are -r 100, -f 180, and -s 18, so this works out to a maximum overlap of 70 bp. If --max-overlap is specified, then the specified value overrides the calculated value. If you do not know the standard deviation of the fragment library, you can probably assume that the standard deviation is 10% of the average fragment length. --interleaved-input Instead of requiring files MATES_1.FASTQ and MATES_2.FASTQ, allow a single file MATES.FASTQ that has the paired-end reads interleaved. Specify "-" to read from standard input. --interleaved-output Write the uncombined pairs in interleaved format. -I, --interleaved Equivalent to specifying both --interleaved-input and --interleaved-output. ******************************** -o, --output-prefix=PREFIX Prefix of output files. Default: "out". -d, --output-directory=DIR Path to directory for output files. Default: current working directory. -c, --to-stdout Write the combined reads to standard output; do not write uncombined reads to anywhere. -z, --compress Compress the FASTQ output files directly with zlib. Similar to specifying --compress-prog=gzip and --suffix=gz, but may be slightly faster. --compress-prog=PROG Pipe the output through the compression program PROG, which will be called as `PROG -c -', plus any arguments specified by --compress-prog-args. PROG must read uncompressed data from standard input and write compressed data to standard output. Examples: gzip, bzip2, xz, pigz. --compress-prog-args=ARGS A string of arguments that will be passed to the compression program if one is specified with --compress-prog. Note: the argument -c is already assumed. --suffix=SUFFIX, --output-suffix=SUFFIX Use SUFFIX as the suffix of the output files after ".fastq". A dot before the suffix is assumed, unless an empty suffix is provided. Default: nothing; or 'gz' if -z is specified; or PROG if --compress-prog is specified. -t, --threads=NTHREADS Set the number of worker threads. This is in addition to the I/O threads. Default: number of processors. Note: if you need FLASH's output to appear deterministically or in the same order as the original reads, you must specify -t 1 (--threads=1). -q, --quiet Do not print informational messages. (Implied with --to-stdout.) -h, --help Display this help and exit. -v, --version Display version.