Manual
Documentation for Sandy version 0.18
Contents
Usage and option summary
General Syntax
Usage:
$ sandy [options]
or
$ sandy help <command>
or even
$ sandy <command> [options] <FILEs>
where there are basically two options for general help, five main commands
with their own inner options, and a specific help
command for each of the
main commands. See:
Options | Description |
---|---|
-h, –help | brief help message |
-M, –man | full documentation |
Help commands | . |
help | show application or command-specific help |
man | show application or command-specific documentation |
Main commands | . |
genome | simulate genome sequencing |
transcriptome | simulate transcriptome sequencing |
custom | simulate custom sequencing |
quality | manage quality profile database |
expression | manage expression-matrix database |
The genome
command
Use it to generate simulated FASTq-files from a given FASTA-file.
The genome
command sets these default options for a genome sequencing simulation:
- The strand is randomly chosen;
- The number of reads is calculated by the coverage;
- The chromosomes are raffled following a weighted raffle with the sequence length as the bias;
Usage:
$ sandy genome [options] <FILEs>
whose options’ exhaustive list can be consulted by sandy genome -h
or
even sandy help genome
commands.
At least one fasta-file must be given as the <FILEs>
term. The results
will be one or two fastq-files, depending on the sequencing-type option,
-t
, for single-ended or paired-ended reads, and an additional reads-count
file.
Options | Description |
---|---|
-h, –help | brief help message |
-M, –man | full documentation |
-v, –verbose | print log messages |
-p, –prefix | prefix output [default:”out”] |
-o, –output-dir | output directory [default:”.”] |
-i, –append-id | append to the defined template id [Format] |
-I, –id | overlap the default template id [Format] |
-j, –jobs | number of jobs [default:”1”; Integer] |
-z, –gzip | compress output file |
-s, –seed | set the seed of the base generator [default:”time()”; Integer] |
-c, –coverage | fastq-file coverage [default:”8”, Number] |
-t, –sequencing-type | single-end or paired-end reads [default:”paired-end”] |
-q, –quality-profile | illumina sequencing system profiles [default:”hiseq”] |
-e, –sequencing-error | sequencing error rate [default:”0.005”; Number] |
-r, –read-size | the read size [default:”101”; Integer] |
-m, –fragment-mean | the fragment mean size for paired-end reads [default:”300”; Integer] |
-d, –fragment-stdd | the fragment standard deviation size for paired-end reads [default:”50”; Integer] |
Some examples:
The command:
$ sandy genome --verbose --sequencing-type=paired-end --coverage=20 hg38.fa 2> sim.log
or, with an equal effect:
$ sandy genome -v -t paired-end -c 20 hg38.fa 2> sim.log
will produce two FASTq-files (sequencing-type default is “paired-end”), both with a coverage of 20x (coverage default is 8), and a simple text reads-count file in a tab separated fashion.
Note: If you use the option -v
, by default, the log messages will be
directed to the standard error so, in the example above, it was redirected
to a file. Without the -v
option, only errors messages will be printed.
For reproducibility, you can set an integer seed for the random raffles
with the -s
option (seed default is environment time()
value),
for example:
$ sandy genome -s 1220 my_fasta.fa
To simulate reads with a ready database registered specific quality profile other than default’s one, type, for example:
$ sandy genome --quality-profile=hiseq_101 hg19.fa
See the quality profile section to know how you can register a new profile.
The sequence identifier is the first and third line of a FASTq entry
beginning with a @ token, for a read identifier, and a +,
for a quality identifier.
Sandy has the capacity to customize it, with a format string passed by
the user. This format is a combination of literal and escaped characters,
in a similar fashion used in C programming language’s printf
function.
For example, let’s simulate a paired-end sequencing and put into it’s
identifier the read length, read position and mate position:
$ sandy genome -s 123 --id="%i.%U read=%c:%t-%n mate=%c:%T-%N length=%r" hg38.fa
In this case, results would be:
$ sandy genome -s 123 --id="%i.%U read=%c:%t-%n mate=%c:%T-%N length=%r" hg38.fa
==> Into R1
@SR.1 read=chr6:979-880 mate=chr6:736-835 length=100
...
==> Into R2
@SR.1 read=chr6:736-835 mate=chr6:979-880 length=100
...
To change the sequencing quality profile, use the -q
option and a
string value (quality-profile default is “hiseq”):
$ sandy genome -q hiseq2 my_fasta_file.fa
You can set the size of the reads with the -r
option and an integer
number (reads-size default is 101):
$ sandy genome -r 151 my_fasta_file.fa
You can set the mean size of a fragment in a paired-end sequencing with
the -m
option and an integer number (default is 300):
$ sandy genome -m 300 my_fasta_file.fa
And you can also set the standard deviation of the size of a fragment in
a paired-end sequencing with the -d
option and an integer number
(default is 50):
$ sandy genome -d 30 my_fasta_file.fa
The options above are the most frequently used ones for the genome
command, but many more can be found in the Sandy’s documentation.
The transcriptome
command
Use it to generate simulated FASTq files from a given FASTA file,
according to an expression profile matrix file.
The transcriptome
command sets these default options for a transcriptome
sequencing simulation as well:
- Choose the Minus strand;
- The number of reads is directly passed;
- The genes/transcripts are raffled following the expression matrix;
Usage:
$ sandy transcriptome [options] <FILEs>
whose options’ exhaustive list can be consulted by sandy transcriptome -h
or
even sandy help transcriptome
commands.
Options | Description |
---|---|
-h, –help | brief help message |
-M, –man | full documentation |
-v, –verbose | print log messages |
-p, –prefix | prefix output [default:”out”] |
-f, –expression-matrix | set the expression matrix [default: none] |
-o, –output-dir | output directory [default:”.”] |
-i, –append-id | append to the defined template id [Format] |
-I, –id | overlap the default template id [Format] |
-j, –jobs | number of jobs [default:”1”; Integer] |
-z, –gzip | compress output file |
-s, –seed | set the seed of the base generator [default:”time()”; Integer] |
-n, –number-of-reads | set the number of reads [default:”1000000”, Integer] |
-t, –sequencing-type | single-end or paired-end reads [default:”paired-end”] |
-q, –quality-profile | illumina sequencing system profiles [default:”hiseq”] |
-e, –sequencing-error | sequencing error rate [default:”0.005”; Number] |
-r, –read-size | the read size [default:”101”; Integer] |
-m, –fragment-mean | the fragment mean size for paired-end reads [default:”300”; Integer] |
-d, –fragment-stdd | the fragment standard deviation size for paired-end reads [default:”50”; Integer] |
Some examples:
The command:
$ sandy transcriptome --verbose --number-of-reads=1000000 --expression-matrix=brain_cortex gencode_pc_v26.fa.gz
or, equivalently
$ sandy transcriptome -v -n 1000000 -f brain_cortex gencode_pc_v26.fa.gz
will generate a FASTq file with 1000000 reads on the gencode_pc_v26.fa.gz file and a plain text file with the raw counts of the reads per gene, according to the expression matrix provided by the brain_cortex entry.
To demonstrate some other features, think about the sequencing error rate that can be set between 0 and 1. By default, Sandy set this value to 0.005, which means 1 error every 200 bases. To set it to another value, try:
$ sandy transcriptome -f liver --sequencing-error=0.001 genome_pc_v26.fa.gz
For reproducibility, the user can set the seed
option and guarantee
the reliability of all the raffles in a later simulation.
$ sandy transcriptome -q hiseq_101 --seed=123 transcripts.fa
The custom
command
This is the most versatile command to produce FASTq-files, but the user must deal whit a greater number of options.
Usage:
$ sandy custom [options] <FILEs>
whose options’ exhaustive list can be consulted by sandy custom -h
or
even sandy help custom
commands.
Options | Description |
---|---|
-h, –help | brief help message |
-M, –man | full documentation |
-v, –verbose | print log messages |
-p, –prefix | prefix output [default:”out”] |
-o, –output-dir | output directory [default:”.”] |
-i, –append-id | append to the defined template id [Format] |
-I, –id | overlap the default template id [Format] |
-j, –jobs | number of jobs [default:”1”; Integer] |
-z, –gzip | compress output file |
-s, –seed | set the seed of the base generator [default:”time()”; Integer] |
-c, –coverage | fastq-file coverage [default:”8”, Number] |
-n, –number-of-reads | directly set the number of reads [Integer] |
-t, –sequencing-type | single-end or paired-end reads [default:”paired-end”] |
-q, –quality-profile | illumina sequencing system profiles [default:”hiseq”] |
-e, –sequencing-error | sequencing error rate [default:”0.005”; Number] |
-r, –read-size | the read size [default:”101”; Integer] |
-m, –fragment-mean | the mean size fragments for paired-end reads [default:”300”; Integer] |
-d, –fragment-stdd | the standard deviation for fragment sizes [default:”50”; Integer] |
-b, –strand-bias | which strand to be used: plus, minus and random [default:”random”] |
-w, –seqid-weight | seqid raffle type: length, same, file [default: “length”] |
-f, –expression-matrix | an expression-matrix entry from database, when seqid-weight=count |
Some examples
The custom
command is the most versatile one, it’s design was thought
to bring user’s with the most of the options between genome
and
transcriptome
commands in a unique command. To have an idea of it’s
plurality, look to how overwhelming the number of choices could be:
$ sandy transcriptome \
--expression-matrix=pancreas \
--quality-profile=hiseq_101 \
--sequencing-type=paired-end \
--fragment-mean=350 \
--fragment-stdd=100 \
--prefix=pancreas_sim \
--output-dir=sim_dir \
--id="%i.%U read=%c:%t-%n mate=%c:%T-%N length=%r" \
--verbose \
--seed=123 \
--jobs=30 \
--no-gzip \
gencode_pc_v26.fa.gz
A note on parallelism: To increase the processing speed, the simulation can run in parallel, splitting the task among jobs. For example, type:
$ sandy custom -f testis -q hiseq_101 -v -i "length=%r" --jobs 15 gencode_lnc.fa.gz
and Sandy will allocate 15 jobs. This feature works for the genome
and
the transcriptome
commands as well.
The quality
command
Use it to manage your quality profile database. You can add or remove your own expression profiles in the builtin database and turn your simulations more realistic based on real experimental data. Or you can even clean it up to restore the vendor’s original entries state. By default, Sandy uses a Poisson distribution when compiling the quality entries, but like many other features, this behavior can be overridden by the user.
Usage:
$ sandy quality [options] <sub-command> <FILEs>
whose options’ exhaustive list can be consulted by sandy quality -h
or
even sandy help quality
commands.
Options | Description |
---|---|
-h, –help | brief help message |
-M, –man | full documentation |
Sub-commands | . |
add | add a new quality profile to database |
remove | remove an user quality profile from database |
restore | restore the database |
Some examples:
To list the quality profiles already registered in the builtin database, you can simply type:
$ sandy quality
and all entries will be shown.
So, to register a new probabilistic quality profile, called, for example, ‘my_profile.txt’, to be used in the simulation of your FASTA-file. You can type:
$ sandy quality add my_profile.txt
This quality profile can be either a FASTq file or a plain text file in a tab separated fashion (quality profile default density function is “Poisson”).
Note: Before the new entry can appear in the database’s list, the new profile needs to be validated, and if it can’t, an error message will be show. Sandy prevents you before overwrite an existing entry.
Sometimes you will need to update or delete some quality profile entry (‘my_profile.txt’ for example) in the database. In this situation, you can remove some actual entry and register a newer one, like this:
$ sandy quality remove my_profile.txt
Sandy will refuse to remove any vendor’s original entry from the database.
And, there could be times when you would want to reset all the database to its original state. It’s a very simple command:
$ sandy quality restore
Note that this is a dangerous command and Sandy will warn you about it before make the restoration in fact.
Note: Sandy already comes with one quality profile based on the Poisson probabilistic curve, as described by the literature (illumina, 2018).
The expression
command
The expression
command is used to verify and update the expression matrix
database. In a transcriptome sequencing simulation, the user
must provide an expression matrix indexed into this database. Sandy
already comes with 52 different tissues from the GTEx project, but the
user has the freedom to include his own data as well, or even clean it up
to restore the vendor’s original entries state.
Usage:
$ sandy expression <sub-command> [options] <FILEs>
whose options’ and sub-commands’ exhaustive list can be consulted by
sandy expression -h
or even sandy help expression
commands.
Options | Description |
---|---|
-h, –help | brief help message |
-M, –man | full documentation |
sub-commands | . |
add | add a new expression-matrix to database |
remove | remove an user expression-matrix from database |
restore | restore the database |
Some examples:
To list the expression matrices already registered in the builtin database, you can simply type:
$ sandy expression
and all registered entries will be shown.
But, suppose you want to register a new expression matrix file called ‘my_mtx.txt’, to simulate your FASTA-file according to its experimentally annotated data. In this case, the command bellow would solve your problem:
$ sandy expression add my_mtx.txt
Note that before the new entry can appear in the database’s list, the new matrix file needs to be validated, and if it can’t, an error message will be show. Sandy prevents you before overwrite an existing entry.
Sometimes you will need to update or delete some expression-matrix entry (‘my_mtx.txt’ for example) in the database. In this situation, you can remove the actual entry and register a newer one, like this:
$ sandy expression remove my_mtx.txt
Sandy will refuse to remove any vendor’s original entry from the database.
And, there could be times when you would want to reset all the database to its original state. It’s a very simple command:
$ sandy expression restore
Note that this is a dangerous command and Sandy will warn you about it before make the restoration in fact.
The help
command
Usage:
To get a simple general help, you can type any of these commands:
$ sandy --help
or for short
$ sandy -h
or simply call it without any arguments.
$ sandy
But, if you need a more comprehensive explanation, you can invoke Sandy’s manual:
$ sandy --man
or for short
$ sandy -M
For help about specific commands, its options and inputs, type:
$ sandy help <command>
or
$ sandy <command> -h
And you can always get help by consulting Sandy’s manuals in your system’s
builtin documentations with man sandy
or info sandy
commands.