ROCKHOPPER(1)

💖 ROCKHOPPER(1) 💖

okay bestie, welcome to rockhopper — the command line tool that lives in the General Commands Manual section of your man pages (section 1, iykyk). It's a whole system for analyzing bacterial RNA-seq data and honestly? she's iconic. Let's get into it. ✨

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✨ NAME ✨

rockhopper — a system for analyzing bacterial RNA-seq data (command line tool) 🦋

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✨ SYNOPSIS ✨

rockhopper [options]

that's it. that's the whole vibe. options go in, science comes out. 💅

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✨ DESCRIPTION ✨

So like, rockhopper is a comprehensive and user-friendly system for computational analysis of bacterial RNA-seq data. no cap. 🧬

As input, it takes RNA sequencing reads output by high-throughput sequencing technology — and those can be FASTQ, QSEQ, FASTA, SAM, or BAM files. any of those five, she's flexible like that. 🎀

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🌸 REQUIRED ARGUMENTS 🌸

these are not optional, bestie, you HAVE to give her these:

• exp1A.fastq,exp1B.fastq,exp1C.fastq exp2A.fastq,exp2B.fastq — a comma separated list of sequencing files (in FASTQ, QSEQ, FASTA, SAM, or BAM format) for replicate experiments, one list per experimental condition. and listen: mate-pair files should be delimited by %. don't forget the %, I'm begging you. 🫶

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🦋 REFERENCE BASED ASSEMBLY VS. DE NONO ASSEMBLY 🦋

okay so this is the big fork in the road, main character decision time:

If the -g option is used, then rockhopper aligns reads to one or more reference genomes. Otherwise, rockhopper performs de novo transcript assembly. two completely different journeys, same girl. ✨

• -g <DIR1,DIR2> — a comma separated list of directories, each containing a genome file (*.fna), gene file (*.ptt), and rna file (*.rnt). yes, all three. she's thorough. 💅

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💜 OPTIONAL ARGUMENTS FOR EITHER REFERENCE BASED ASSEMBLY OR DE NOVO ASSEMBLY 💜

these work no matter which path you picked, so they're like the universal besties:

• -c <boolean> — reverse complement single-end reads (default is false)
• -ff, -fr, -rf, -rr — orientation of two mate reads for paired-end read, f=forward and r=reverse_complement (default is fr)
• -d <integer> — maximum number of bases between mate pairs for paired-end reads (default is 500)
• -a <boolean> — identify 1 alignment (true) or identify all optimal alignments (false), (default is true)
• -p <integer> — number of processors (default is self-identification of processors, she'll figure it out herself, independent queen 👑)
• -e <boolean> — compute differential expression for transcripts in pairs of experimental conditions (default is true)
• -s <boolean> — RNA-seq experiments are strand specific (true) or strand ambiguous (false), (default is true)
• -L <comma separated list> — labels for each condition
• -o <DIR> — directory where output files are written (default is Rockhopper_Results/)
• -v <boolean> — verbose output including raw/normalized counts aligning to each gene (default is false)
• -SAM — output a SAM format file
• -TIME — output time taken to execute program ⏱️

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🎀 OPTIONAL ARGUMENTS FOR REFERENCE BASED ASSEMBLY ONLY 🎀

these ones only show up when you're doing the reference based thing (so, when -g is in the building):

• -m <number> — allowed mismatches as percent of read length (default is 0.15)
• -l <number> — minimum seed as percent of read length (default is 0.33)
• -y <boolean> — compute operons (default is true)
• -t <boolean> — identify transcript boundaries including UTRs and ncRNAs (default is true)
• -z <number> — minimum expression of UTRs and ncRNAs, a number in range [0.0, 1.0] (default is 0.5)

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🌈 OPTIONAL ARGUMENTS FOR DE NOVO ASSEMBLY ONLY 🌈

and these ones are de novo assembly exclusive, the de novo girlies only:

• -k <integer> — size of k-mer, range of values is 15 to 31 (default is 25)
• -j <integer> — minimum length required to use a sequencing read after trimming/processing (default is 35)
• -n <integer> — size of k-mer hashtable is ~ 2^n (default is 25).

HINT: should normally be 25 or, if more memory is available, 26. WARNING: if increased above 25 then more than 1.2M of memory must be allocated. ⚠️ like, actually allocate it. she will not be okay otherwise. 💀

• -b <integer> — minimum number of full length reads required to map to a de novo assembled trancript (default is 20)
• -u <integer> — minimum length of de novo assembled transcripts (default is 2*k)
• -w <integer> — minimum count of k-mer to use it to seed a new de novo assembled transcript (default is 50)
• -x <integer> — minimum count of k-mer to use it to extend an existing de novo assembled transcript (default is 5)

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⭐ EXAMPLES ⭐

here's where it gets real. copy these, they're free. 🍓

reference based assembly with single-end reads

% rockhopper <options> -g genome_DIR1,genome_DIR2 aerobic_replicate1.fastq,aerobic_replicate2.fastq anaerobic_replicate1.fastq,anaerobic_replicate2.fastq

de novo assembly with single-end reads

% rockhopper <options> aerobic_replicate1.fastq,aerobic_replicate2.fastq anaerobic_replicate1.fastq,anaerobic_replicate2.fastq

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🔥 SEE ALSO 🔥

• https://cs.wellesley.edu/~btjaden/Rockhopper/
• rockhoppergui(1)

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and that's the whole page, bestie!! 🫶 this one comes from the February 2022 era of ROCKHOPPER(1) — go forth, assemble some transcripts, and remember: reference based if you got a -g, de novo if you don't. slay. 💖✨🎀🦋