Sailfish¶
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 needs to be run once 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.
Indexing¶
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.
Quantification¶
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 fragment (read) library (see Fragment Library Types), <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.