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At default of 0. All others receive no strand call e. Only stranded loci are analyzed for MIRNAs, while only unstranded loci are analyzed with respect to phasing.
Most users probably want to use the default setting of 0. Specifying this value here speeds the analysis, since ShortStack does not need to count the reads directly from the bam file.
Can only be specified in conjunction with --bamfile. At least 4G memory is suggested. Alignment and building bowtie indices tend to be the most memory-intensive portions for a given run, and memory usage seems to scale with genome size, but not as much with the number of small RNAs.
All other portions are single-threaded. At least 50G of hard disk space is recommended to be available, due to the sometimes large size of the temporary alignment files and the final alignment file.
The total time of analysis depends on several factors, including most prominently genome size, number of reads analyzed, whether or not bowtie indices need to be created, whether or not MIRNAs are being analyzed, and of course your equipment.
Specifying --readfile is mutually exclusive with both --bamfile or --cramfile Details of alignment methods and performance testing For full details on ShortStack's alignment methods and the results of performance testing, see Johnson et al.
Genome pre-processing Genome file format and naming All runs require a reference genome in FASTA format, specified with the --genomefile option.
The file must end with a valid suffix.. Within the genome, if the name of a chromosome has whitespace characters, the name will be trimmed at the first whitespace character.
This can drastically improve performance during MIRNA searching for highly fragmented genome assemblies. As of ShortStack 3.
Genome indexing If not detected, an index of the genome will be created using samtools faidx. This can be time-consuming, and memory intensive.
Reads pre-processing Reads file formats Small RNA reads to be aligned must be in fasta, fastq, or csfasta formats, or their gzip-compressed versions.
File names must end with. Colorspace reads cannot be mixed with base-space reads; otherwise, mixed file formats are ok. Color-space quality values are NOT accepted in.
No paired-end support There is no support for paired-end reads in ShortStack. No condensation Input reads are expected to be de-condensed.
That is, if a small RNA was sequenced 10, times in a run, there should be 10, entries, each with a different header name, in the input readfile.
In other words, ShortStack is designed to take reads right off the sequencer without any other pre-processing except adapter trimming.. Unique read names required The small RNA reads must all have unique names within a given file.
If this requirement is not met, alignments will be completely unreliable due to errors in interpreting and handling of multi-mapped reads.
Adapter trimming ShortStack has a primitive 3'-adapter capability. Specify an adapter of at least 8nts in length with option --adapter.
If nothing is given to --adapter, ShortStack assumes your reads are already trimmed. Trimming simply looks for the right-most exact match to the given apdater sequence, and when found, chops it off.
If a read is smaller than 15nts after trimming, it is discarded. For more sophisticated adapter trimming, consider cutadapt or trimmomatic If quality values are present, they are trimmed as well.
Alignment overview ShortStack uses bowtie to align reads. It first aligns, and processes the output on the fly to note how many equally good alignment positions were found for each read.
It then uses this information in a second phase to 'decide' on the most likely 'correct' location for multi-mapped reads. The final output is a single.
If multiple readfiles were input, the final bam or cram file notes the origin of each read with the RG tag see sam format specification.
This helps with sequencing errors and SNPs. If a read has some alignments with 0 mismatches, and some with 1, only those with 0 mismatches are kept. The option --mismatches controls this threshold, and can be set to 0, 1, or 2.
To get around this, when aligning to a 'large' reference, ShortStack forces the number of allowed mismatches to be 0.
The default setting is The choice of method is specified with the option --mmap. The methods are: u: Placement guided by uniquely mapping reads.
During the alignment, the count of uniquely mapped reads is kept in 50nt bins across the reference genome. The bin location is determined by the left-most coordinate of the uniquely mapped read.
After the first phase of alignment for all reads in all files has completed, this genome-wide map of uniquely-mapped read counts is used to guide the decisions of the most likely locations of multi-mapped reads.
Specifically, for a given multi-mapped read, the local count of uniquely mapped reads at each possible location is computed. The local count is that of the specific 50nt bin the alignment lies in again, by left-most positon plus the counts of the 2 bins upstream and 2 bins downstream.
All of the local counts are converted to fractions of the sum of all total counts. These fractions are then used as the probabilities of placement for the multi-mapped read.
For instance, suppose a multi-mapped read had three possible positions. The read counts of uniquely mapped reads were 30, 65, and 5.
The actual choice is probabilistic, given the computed weightings, for each read. Like u, except that multi-mapped reads also contribute to the guidance densities.
This is faster than u and f, but performs much more poorly at properly placing multi-mapped reads. Achieves high sensitivity, but very low precision.
Very fast, but ignores large quantities of data. Achieves high precision, but very low sensitivity. The default setting for --mmap is u ranmax When running mmap method u or f, there are some cases where no guidance can be given, and so the choice on where to put a multi-mapped read is still random.
In those cases, the option ranmax will suppress any alignment where the choice is 'too' random. By default, --ranmax is set at 3, so that if a read can't be placed confidently, no placement is done if there are more than 3 choices.
Alignment output format Final alignments are sorted bam or cram formatted alignments. XX:i tags: Added by ShortStack to each line, this indicates the total number of valid alignments found for the read.
XZ:f tags: Added by ShortStack to each line, this indicates the calculated probability of placement for the read.
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