• A C++-11 compliant version of GCC. Any version of g++ >= 4.7 should work.

• CMake. Sailfish uses the CMake build system to check, fetch and install dependencies, and to compile and install Sailfish. CMake is available for all major platforms (though Sailfish is currently unsupported on Windows.)

After downloading the Sailfish source distribution and unpacking it, change into the top-level directory:

> cd Sailfish-0.6.3-Source

Then, create an out-of-source build directory and change into it:

> mkdir build
> cd build

Sailfish makes extensive use of Boost. We recommend installing the most recent version (1.55) systemwide if possible. If Boost is not installed on your system, the build process will fetch, compile and install it locally. However, if you already have a recent version of Boost available on your system, it make sense to tell the build system to use that.

If you have Boost installed you can tell CMake where to look for it. Likewise, if you already have Intel's Threading Building Blocks library installed, you can tell CMake where it is as well. The flags for CMake are as follows:

• -DFETCH_BOOST=TRUE -- If you don't have Boost installed (or have an older version of it), you can provide the FETCH_BOOST flag instead of the BOOST_ROOT variable, which will cause CMake to fetch and build Boost locally.

• -DBOOST_ROOT= -- Tells CMake where an existing installtion of Boost resides, and looks for the appropritate version in . This is the top-level directory where Boost is installed (e.g. /opt/local).

• -DTBB_INSTALL_DIR= -- Tells CMake where an existing installation of Intel's TBB is installed (), and looks for the apropriate headers and libraries there. This is the top-level directory where TBB is installed (e.g. /opt/local).

• -DCMAKE_INSTALL_PREFIX=<install_dir> -- <install_dir> is the directory to which you wish Sailfish to be installed. If you don't specify this option, it will be installed locally in the top-level directory (i.e. the directory directly above "build").

Setting the appropriate flags, you can then run the CMake configure step as follows:

> cmake [FLAGS] ..

The above command is the cmake configuration step, which should complain if anything goes wrong. Next, you have to run the build step. Depending on what libraries need to be fetched and installed, this could take a while (specifically if the installation needs to install Boost). To start the build, just run make.

> make

If the build is successful, the appropriate executables and libraries should be created. There are two points to note about the build process. First, if the build system is downloading and compiling boost, you may see a large number of warnings during compilation; these are normal. Second, note that CMake has colored output by default, and the steps which create or link libraries are printed in red. This is the color chosen by CMake for linking messages, and does not denote an error in the build process.

Finally, after everything is built, the libraries and executable can be installed with:

> make install

To ensure that Sailfish has access to the appropriate libraries you should ensure that the PATH variabile contains <install_dir>/bin, and that LD_LIBRARY_PATH (or DYLD_FALLBACK_LIBRARY_PATH on OSX) contains <install_dir>/lib.

After the paths are set, you can test the installation by running

> make test

This should run a simple test and tell you if it succeeded or not.

Sailfish is a tool for transcript quantification from RNA-seq data. It requires a set of target transcripts (either from a reference or de-novo assembly) to quantify. All you need to run Sailfish is a fasta file containing your reference transcripts and a (set of) fasta/fastq file(s) containing your reads. Sailfish runs in two phases; indexing and quantification. The indexing step is independent of the reads, and only need to be run one for a particular set of reference transcripts and choice of k (the k-mer size). The quantification step, obviously, is specific to the set of RNA-seq reads and is thus run more frequently. For a more complete description of all available options in Sailfish, see the manual.

To generate the Sailfish index for your reference set of transcripts, you should run the following command:

> sailfish index -t <ref_transcripts> -o <out_dir> -k <kmer_len>

This will build a Sailfish index for k-mers of length <kmer_len> for the reference transcripts provided in the file <ref_transcripts> and place the index under the directory <out_dir>. There are additional options that can be passed to the Sailfish indexer (e.g. the number of threads to use). These can be seen by executing the command "Sailfish index -h".

Now that you have generated the Sailfish index (say that it's the directory <index_dir> --- this corresponds to the <out_dir> argument provided in the previous step), you can quantify the transcript expression for a given set of reads. To perform the quantification, you run a command like the following:

> sailfish quant -i <index_dir> -l "<libtype>" {-r <unmated> | -1 <mates1> -2 <mates2>} -o <quant_dir>

Where <index_dir> is, as described above, the location of the sailfish index, <libtype> is a string describing the format of the read library (see the library string section below) <unmated> is a list of files containing unmated reads, <mates{1,2}> are lists of files containg, respectively, the first and second mates of paired-end reads. Finally, <quant_dir> is the directory where the output should be written. Just like the indexing step, additional options are available, and can be viewed by running "sailfish quant -h".

When the quantification step is finished, the directory <quant_dir> will contain a file named "quant.sf" (and, if bias correction is enabled, an additional file names "quant_bias_corrected.sf"). This file contains the result of the Sailfish quantification step. This file contains a number of columns (which are listed in the last of the header lines beginning with '#'). Specifically, the columns are (1) Transcript ID, (2) Transcript Length, (3) Transcripts per Million (TPM), (4) Reads Per Kilobase per Million mapped reads (RPKM), (5) K-mers Per Kilobase per Million mapped k-mers (KPKM), (6) Estimated number of k-mers (an estimate of the number of k-mers drawn from this transcript given the transcript's relative abundance and length) and (7) Estimated number of reads (an estimate of the number of reads drawn from this transcript given the transcript's relative abnundance and length). The first two columns are self-explanatory, the next four are measures of transcript abundance and the final is a commonly used input for differential expression tools. The Transcripts per Million quantification number is computed as described in [1], and is meant as an estimate of the number of transcripts, per million observed transcripts, originating from each isoform. Its benefit over the K/RPKM measure is that it is independent of the mean expressed transcript length (i.e. if the mean expressed transcript length varies between samples, for example, this alone can affect differential analysis based on the K/RPKM.) The RPKM is a classic measure of relative transcript abundance, and is an estimate of the number of reads per kilobase of transcript (per million mapped reads) originating from each transcript. The KPKM should closely track the RPKM, but is defined for very short features which are larger than the chosen k-mer length but may be shorter than the read length. Typically, you should prefer the KPKM measure to the RPKM measure, since the k-mer is the most natural unit of coverage for Sailfish.

The library format string is given as a parameter to the quant step of Sailfish. Since Sailfish works with the reads directly and not alignments, the purpose of this string is to inform Sailfish of relevant information about the reads in the library. Not all of this information is currently used, but some of it is and other pieces of it may be in the future.

The library format string consists of up to 3 parts (2 parts for unpaired reads), provided as key-value pairs. The key-value pairs are provided with the syntax "key=value", and they are separated by the : character. The relevant keys and possible value options are:

(T|TYPE)=(PE|SE)

This option specifies the "paired-end" status of the read library. If the reads are paired end, then this should be set to PE, and the library format string should be followed by the -1 and -2 options with the respective mate-pair reads. If the reads are unpaired, then this should be set to SE and the library format string shoujld be followed by the -r option and list of files containing unpaired reads.

(O|ORIENTATION)=(>>|<>|><)

This option secifies the relative orientation of reads within a pair. If the library consists of unpaired reads, then this key-value pair can and should be ignored. If the library consists of paired end reads, this key-value pair should be provided. Note, this denotes the realtive orientation of the reads, not their absolute directionality with respect to the reference. The options are meant to denote, visually, how the reads could be oriented.

The first option >> denotes that the mates are oriented in the same direction --- e.g. if the 5' end of mate 1 is upstream from the 3' end, then the 5' end of mate 2 is upstream from its 3' end and vice-versa.

The second option <> denotes that the mates are oriented away from each other. This implies that start of mate1 is closer to start of mate 2 than the end of mate 2, etc.

The third option >< is, perhaps, the most common relative orientation. It denotes that the mates are oriented toward each other, so that the start of mate 1 is farther from the start of mate 2 than it is from the end of mate 2 and vice-versa.

(S|STRAND)=(AS|SA|S|A|U)

This option specifies the strandedness of the reads. If the type is SE the only allowable options are S, A, and U, which denote, respectively, that the reads come from the sense strand, the antisense strand, or are of unknown strandedness (in which case both strands are tried and the one resulting in more matching k-mers is used).

If the type of the read library is PE, then any of the options are valid. The S, A and U options given in the above paragraph have the same meaning. The AS option specifies that mate 1 is from the antisense strand and mate2 is from the sense strand, while SA specifies that mate 1 is from the sense strand and mate 2 is from the antisense strand.

Because of the way argument parsing works, the library format string must be offset by quotations. An example format string specifying that the read library consists of unpaired reads with unknown orientation is:

-l "T=SE:S=U"

Alternatively, a format string specifying that the read library consists of paired-end reads, oriented toward each other where mate 1 is from the sense strand and mate 2 is from the antisense strand is:

-l "T=PE:O=><:S=SA"

Many, but not all, combinations of the three options (type, orientation and strandedness) are possible. Sailfish will perform a coarse-grained sainty check to ensure that the provided library string is not impossible (e.g. T=SE:O=><:S=A, which is not possible because unpaired reads can't have a relative orientation).

This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version.

This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.

You should have received a copy of the GNU General Public License along with this program. If not, see <http://www.gnu.org/licenses/\>.

[1] Li, Bo, et al. "RNA-Seq gene expression estimation with read mapping uncertainty." Bioinformatics 26.4 (2010): 493-500.