FLASH(1)

💅 FLASH(1) — User Commands

okay so the manual name is FLASH and it lives in section 1 (User Commands), so if you went looking for man flash, bestie you are exactly where you need to be 💖

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✨ NAME ✨

flash — Fast Length Adjustment of SHort reads

🦋 SYNOPSIS 🦋

flash [OPTIONS] MATES_1.FASTQ MATES_2.FASTQ

flash [OPTIONS] --interleaved-input (MATES.FASTQ | -)

flash [OPTIONS] --tab-delimited-input (MATES.TAB | -)

🌸 DESCRIPTION 🌸

FLASH (Fast Length Adjustment of SHort reads) is an accurate and fast little icon that merges paired-end reads made from DNA fragments whose lengths are shorter than twice the length of the reads. When you merge read pairs you get unpaired longer reads, and those are generally way more wanted in genome assembly and genome analysis processes. it's giving glow-up, honestly.

So briefly — and this is the part where FLASH is lowkey a genius — the FLASH algorithm considers all possible overlaps at or above a minimum length between the reads in a pair, and then picks the overlap that results in the lowest mismatch density (that's the proportion of mismatched bases in the overlapped region). If there's a tie between multiple overlaps, it breaks the tie by looking at the quality scores at the mismatch sites. And when it's building the merged sequence, FLASH computes a consensus sequence in the overlapped region. more deets live in the original publication (http://bioinformatics.oxfordjournals.org/content/27/21/2957.full).

Limitations of FLASH include: 🙈

• FLASH cannot merge paired-end reads that do not overlap. like, at all. no overlap no merge, periodt.
• FLASH is not designed for data that has a significant amount of indel errors (such as Sanger sequencing data). It is best suited for Illumina data.

🌈 MANDATORY INPUT 🌈

The most common input to FLASH is two FASTQ files, holding read 1 and read 2 of each mate pair respectively, in the same order. order matters bestie, don't come in here shuffling things.

Alternatively you may provide one FASTQ file — which may be standard input — containing paired-end reads in either interleaved FASTQ (peep the --interleaved-input option) or tab-delimited (peep the --tab-delimited-input option) format. In all cases, gzip compressed input is autodetected. Also, in all cases, the PHRED offset is, by default, assumed to be 33; use the --phred-offset option to change it.

👑 OUTPUT 👑

The default output of FLASH consists of the following files:

• out.extendedFrags.fastq — the merged reads ✨
• out.notCombined_1.fastq — read 1 of mate pairs that were not merged
• out.notCombined_2.fastq — read 2 of mate pairs that were not merged
• out.hist — numeric histogram of merged read lengths
• out.histogram — visual histogram of merged read lengths

FLASH also logs informational messages to standard output. These can also be redirected to a file, as in the following example:

$ flash reads_1.fq reads_2.fq 2>&1 | tee flash.log

In addition, FLASH supports several features affecting the output:

• Writing the merged reads directly to standard output (--to-stdout)
• Writing gzip compressed output files (-z) or using an external compression program (--compress-prog)
• Writing the uncombined read pairs in interleaved FASTQ format (--interleaved-output)
• Writing all output reads to a single file in tab-delimited format (--tab-delimited-output)

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💖 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 65bp, 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: 65bp, 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.
• -O, --allow-outies — also try combining read pairs in the "outie" orientation, e.g.

Read 1: <-----------
       Read 2:       ------------>

as opposed to only the "innie" orientation, e.g.

Read 1:
       <------------

Read 2: ----------->

FLASH uses the same parameters when trying each orientation. If a read pair can be combined in both "innie" and "outie" orientations, the better-fitting one will be chosen using the same scoring algorithm that FLASH normally uses.

This option also causes extra .innie and .outie histogram files to be produced.

• -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 65 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.

• --cap-mismatch-quals — cap quality scores assigned at mismatch locations to 2. This was the default behavior in FLASH v1.2.7 and earlier. Later versions will instead calculate such scores as max(|q1 - q2|, 2); that is, the absolute value of the difference in quality scores, but at least 2. Essentially, the new behavior prevents a low quality base call that is likely a sequencing error from significantly bringing down the quality of a high quality, likely correct base call.
• --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 FASTQ format.
• -I, --interleaved — equivalent to specifying both --interleaved-input and --interleaved-output.
• -Ti, --tab-delimited-input — assume the input is in tab-delimited format rather than FASTQ, in the format described below in '--tab-delimited-output'. In this mode you should provide a single input file, each line of which must contain either a read pair (5 fields) or a single read (3 fields). FLASH will try to combine the read pairs. Single reads will be written to the output file as-is if also using --tab-delimited-output; otherwise they will be ignored. Note that you may specify "-" as the input file to read the tab-delimited data from standard input.
• -To, --tab-delimited-output — write output in tab-delimited format (not FASTQ). Each line will contain either a combined pair in the format tag <tab> seq <tab> qual or an uncombined pair in the format tag <tab> seq_1 <tab> qual_1 <tab> seq_2 <tab> qual_2.
• -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. In this mode, with FASTQ output (the default) the uncombined reads are discarded. With tab-delimited output, uncombined reads are included in the tab-delimited data written to standard output. In both cases, histogram files are not written, and informational messages are sent to standard error rather than to standard output.
• -z, --compress — compress the output files directly with zlib, using the gzip container format. 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 when invoked as noted above. Examples: gzip, bzip2, xz, pigz.
• --compress-prog-args=ARGS — a string of additional arguments that will be passed to the compression program if one is specified with --compress-prog=PROG. (The arguments '-c -' are still passed in addition to explicitly specified arguments.)
• --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=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. 🤫
• -h, --help — display this help and exit.
• -v, --version — display version.

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okay that's this chunk, and bestie there is more page coming so don't go anywhere 💜

💖 AUTHOR 💖

Ok so who actually wrote this masterpiece? This manpage was written by Andreas Tille for the Debian distribution, and it can be used for any other usage of the program. 🎀

That's it. That's the whole section. He wrote it for Debian, but bestie, the door is open — any other usage of the program is totally fine too. Slay, Andreas. 💅✨

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And that's the whole page, bestie!! 🦋 You made it to the end, which honestly? Main character energy. This one's from flash 1.2.11, dated May 2020, so go forth and align your reads with confidence. Periodt. 💖🌸👑