sideRETRO has a very straightforward syntax. Basically, there are three main commands, each one with a plethora of available options:
process-sample
merge-call
make-vcf
So, in order to test the installation process and run a first example, user can call it without any argument from the command line, like this:
$ sider Usage: sider [-hv] sider <command> [options] A pipeline for detecting Somatic Insertion of DE novo RETROcopies Options: -h, --help Show help options -v, --version Show current version -c, --cite Show citation in BibTeX Commands: ps, process-sample Extract alignments related an event of retrocopy mc, merge-call Discover and annotate retrocopies vcf, make-vcf Generate VCF file with all annotate retrocopies
In the above situation, if sideRETRO was correctly installed, it will give that default usage help.
Another classical example is to print sideRETRO's installed version using
the -v
option:
$ sider --version sideRETRO 1.0.0
And, if the user need further help, he can find it both at the sideRETRO's readthedocs page or in the already installed software documentation, from command line:
$ sider --help
Please, see :ref:`A Practical Workflow <pract_wf>` and :ref:`Running with Docker <run_dck>` sections for more examples and tips for using with Docker.
Now, to get more familiar with sideRETRO main commands and results, let's try some basic examples for each command.
The first one is process-sample
or ps
for short, and was intended to act
as the "evidence's grounding faith" for sideRETRO. Here, we're saying
"first" because of an order in which the user must run the commands. The file
resultant from this command will become the input to the next one,
merge-call
.
As explained in the Introduction section, the command
process-sample
creates a database of abnormal reads from a SAM/BAM file set.
To do this, there are some mandatory options the user must supply to do a
correct search. Calling the command process-sample
without any argument
will give a specific help where user can know all the mandatory options for
this command:
$ sider process-sample
- Arguments:
- One or more alignment file in SAM/BAM format
- Mandatory Options:
-a, --annotation-file Gene annotation on the reference genome in GTF/GFF3 format. sider will look for 'exon' with the attribute 'transcript_type=protein_coding'. The attributes 'gene_name', 'gene_id' and 'exon_id' are also required -i, --input-file File containing a newline separated list of alignment files in SAM/BAM/CRAM format. This option is not mandatory if one or more SAM/BAM/CRAM files are passed as argument. If 'input-file' and arguments are set concomitantly, then the union of all alignment files is used - Input/Output Options:
-h, --help Show help options -q, --quiet Decrease verbosity to error messages only or suppress terminal outputs at all if 'log-file' is passed --silent Same as '--quiet' -d, --debug Increase verbosity to debug level -l, --log-file Print log messages to a file -o, --output-dir Output directory. Create the directory if it does not exist [default:"."] -p, --prefix Prefix output files [default:"out"] - SQLite3 Options:
-c, --cache-size Set SQLite3 cache size in KiB [default:"200000"] - Read Quality Options:
-Q, --phred-quality Minimum mapping quality of the reads required [default:"8"] -M, --max-base-freq Maximum base frequency fraction allowed [default:"0.75"] -D, --deduplicate Remove duplicated reads. Reads are considered duplicates when they share the 5 prime positions of both reads and read-pairs - Processing Options:
-s, --sorted Assume all reads are grouped by queryname, even if there is no SAM/BAM/CRAM header tag 'SO:queryname' -t, --threads Number of threads [default:"1"] -m, --max-distance Maximum distance allowed between paired-end reads [default:"10000"] -f, --exon-frac Minimum overlap required as a fraction of exon [default:"1e-09"; 1 base] -F, --alignment-frac Minimum overlap required as a fraction of alignment [default:"1e-09"; 1 base] -e, --either The minimum fraction must be satisfied for at least exon OR alignment. Without '-e', both fractions would have to be satisfied -r, --reciprocal The fraction overlap must be reciprocal for exon and alignment. If '-f' is 0.5, then '-F' will be set to 0.5 as well
So, supposing that the user has three files: f1.bam, f2.bam, f3.sam, he can type:
$ sider process-sample f2.bam f2.bam f3.sam \ -a annotation_file.gtf
Note the mandatory -a
option specifying the annotation file. And, in this
unique exception, we suppressed the -i
mandatory option cause all the files
were explicitly called.
Let's see another example that shows the convenient use of the -i
option to
call a list of input files (e.g. my_files_list.txt) instead of them directly:
$ sider process-sample \ -i my_files_list.txt \ -a annotation_file.gtf
Both commands above will produce only one output database file out.db
containing all relevant reads for non-fixed retrocopies search, whose prefix
out can be easily changed with the -p
option. The abnormal reads from
all input files will be merged in just one table. To produce one database for
each input file separately, user must run one distinct instance of
sideRETRO per file.
Some options' values can affect drastically the output. Let's play a little bit
with some of them while using the short version of the command ps
:
$ sider ps \ -i my_files_list.txt \ -a annotation_file.gtf \ -o output_dir \ -p my_reads_database \ -l my_log_file.log \ -c 2000000 \ -Q 20 \ -F 0.9 \ -t 3
Wow! The number of options can be overwhelming.
Here used -o
option to specify the directory output_dir to write our
database as my_reads_database.db (-p
option). Also, we chose to save the
log messages in my_log_file.log file (-l
option), a cache size of 2Gb
(-c
option), a minimum phred score cutoff of 20 for alignments (-Q
option), a minimum overlap ratio of 0.9 for read alignments over exonic regions
(-F
option) and 3 threads to process those files in parallel (-t
option).
To see another example of the process-sample
command chained in a real
workflow, please refer to the :ref:`A Practical Workflow <pract_wf>` section.
The second step in the sideRETRO's "journey for the truth of retrocopies"
is the command merge-call
or mc
for short. The aim of this command is to
take the database created by process-sample
step as input and populate more
tables in it, with information risen from a clustering process over the abnormal
reads regions.
Like process-sample
, merge-call
has some mandatory options, which can be
known by calling it without any argument:
$ sider merge-call
- Arguments:
- One or more SQLite3 databases generated in the process-sample step
- Mandatory Options:
-i, --input-file File containing a newline separated list of SQLite3 databases to be processed. This option is not mandatory if one or more SQLite3 databases are passed as argument. If 'input-file' and arguments are set concomitantly, then the union of all files is used - Input/Output Options:
-h, --help Show help options -q, --quiet Decrease verbosity to error messages only or suppress terminal outputs at all if 'log-file' is passed --silent Same as '--quiet' -d, --debug Increase verbosity to debug level -l, --log-file Print log messages to a file -o, --output-dir Output directory. Create the directory if it does not exist [default:"."] -p, --prefix Prefix output files [default:"out"] -I, --in-place Merge all databases with the first one of the list, instead of creating a new file - SQLite3 Options:
-c, --cache-size Set SQLite3 cache size in KiB [default:"200000"] - Clustering Options:
-e, --epsilon DBSCAN: Maximum distance between two alignments inside a cluster [default:"300"] -m, --min-pts DBSCAN: Minimum number of points required to form a dense region [default:"10"] - Filter & Annotation Options:
-b, --blacklist-chr Avoid clustering from and to this chromosome. This option can be passed multiple times [default:"chrM"] -B, --blacklist-region GTF/GFF3/BED blacklisted regions. If the file is in GTF/GFF3 format, the user may indicate the 'feature' (third column), the 'attribute' (ninth column) and its values -P, --blacklist-padding Increase the blacklisted regions ranges (left and right) by N bases [default:"0"] -T, --gff-feature The value of 'feature' (third column) for GTF/GFF3 file [default:"gene"] -H, --gff-hard-attribute The 'attribute' (ninth column) for GTF/GFF3 file. It may be passed in the format key=value (e.g. gene_type=pseudogene). Each value will match as regex, so 'pseudogene' can capture IG_C_pseudogene, IG_V_pseudogene etc. This option can be passed multiple times and must be true in all of them -S, --gff-soft-attribute Works as 'gff-hard-attribute'. The difference is if this option is passed multiple times, it needs to be true only once [default:"gene_type=processed_pseudogene tag=retrogene"] -x, --parental-distance Minimum distance allowed between a cluster and its putative parental gene [default:"1000000"] -g, --genotype-support Minimum number of reads coming from a given source (SAM/BAM/CRAM) within a cluster [default:"3"] -n, --near-gene-rank Minimum ranked distance between genes in order to consider them close [default:"3"] - Genotyping Options:
-t, --threads Number of threads [default:"1"] -Q, --phred-quality Minimum mapping quality used to define reference allele reads [default:"8"]
And likewise, user can call a set of database files directly, or using a list of files:
$ sider merge-call database1.db database2.db -I
or
$ sider merge-call -i my_databases_list.txt -I
Note
Again, note the -I
option that is not mandatory but would lead the creation
of duplicated output databases if absent. This option do the clustering
"in place" over the input files, overwriting them (so be careful). If user do
not use the -p
or -I
options, the output files will be named out.db.
In a more sophisticated example, we will use the short version of the command
mc
, with many other options:
$ sider mc \ -i my_databases_list.txt \ -o output_dir \ -p my_database \ -l my_log_file.log \ -I \ -c 2000000 \ -B my_black_list.bed \ -x 1000000 \ -g 5 \ -Q 20 \ -C 15 \ -t 3
Here, options -i
, -o
, -p
, -l
, -I
, -c
, -Q
and -t
keeps the same meaning as they have in the process-sample
command.
The others need some explanation. All we've done here was to ask for a minimum
number of 5 reads of contribution from each input SAM/BAM file to consider a
clustering region as a retrocopy candidate (with -g
option); a minimum
distance of 1000000 bp from the parental gene to resolve some doubtful overlaps
(-x
option), a minimum number of 15 crossing reads over the putative
insertion point to consider heterozygosis evidence (-C
) and, importantly,
a BED file with a list of regions to be ignored at the clustering process called
my_black_list.txt (-B
option). This last option's file can describe
entire chromosomes (like chrM) and many chromosomal regions with poor insertion
evidences taken literature, like centromers. All specified regions won't be
targets for clustering.
To see another example of the merge-call
command chained in a real workflow,
please refer to the :ref:`A Practical Workflow <pract_wf>` section.
The third and last step to the sideRETRO's "crusade to retrocopies" is the
make-vcf
command or vcf
for short. This command takes the already
clustered tables in the database files populated at the merge-call
step and
creates one VCF file with all statistically significant retrocopy insertions
annotated in a convenient format.
This command has no mandatory options, but it is worth try to discover the others:
$ sider make-vcf
- Arguments:
- SQLite3 database generated at process-sample and merge-call steps
- Input/Output Options:
-h, --help Show help options -q, --quiet Decrease verbosity to error messages only or suppress terminal outputs at all if 'log-file' is passed --silent Same as '--quiet' -d, --debug Increase verbosity to debug level -l, --log-file Print log messages to a file -o, --output-dir Output directory. Create the directory if it does not exist [default:"."] -p, --prefix Prefix output files [default:"out"] - Filter & Annotation Options:
-n, --near-gene-dist Minimum distance between genes in order to consider them close [default:"10000"] -e, --orientation-error Maximum error allowed for orientation rho [default:"0.05"] -r, --reference-file FASTA file for the reference genome
So, in order to produce a VCF file from a database input file like my_database.db, just type:
$ sider make-vcf my_database.db
This will produce a out.vcf output file.
Let's add more options to customize it to our needs (with the short version of the command only for symmetry):
$ sider vcf my_database.db \ -o output_dir \ -p my_retrocopies \ -l my_log_file.log \ -r my_reference_genome.fa \ -n 50000
Command make-vcf
is very simple and don't allow the user to use threads.
The only new options are -r
, which must specify the reference genome in
FASTA format (like gencode's Hg38.fa) and -n
, where user can establish
a distance threshold for genes surrounding insertion points for additional
information in the output VCF file.
Working with CRAM files may be a little tricky, mainly if you have downloaded the data from a public repository. Let's take a look at two possible cases:
- Local alignment
- External alignment
In order to generate an alignment file in the CRAM format, first we need to index the reference genome:
# Inde for BWA: .fa.amb, .fa.ann, .fa.bwt, .fa.pac, .fa.sa files
bwa index hg38.fa
# Index reference genome for CRAM: .fa.fai file
samtools faidx hg38.fa
Then, we can align with bwa
:
# Align with BWA and generate a CRAM
bwa mem hg38.fa file_R1.fastq file_R2.fastq | \
samtools view -T hg38.fa -C -o file.cram -
The alignment :file:`file.cram` can be processed with sider
, as long as
we don't change the reference genome and its index (:file:`.fa.fai`) path. If so,
we need to set the environment variables :file:`REF_PATH` and :file:`REF_CACHE`,
see :ref:`External alignment <extern_cram>`.
When we download public data already aligned in the CRAM format, we may be
concerned about the reference genome index. Probably, we won't have the
required genome index to read the :file:`.cram`, and the :file:`htslib`
library - used by sider
and samtools
- is able to download
the index from the CRAM Reference Registry.
However, in order to :file:`htslib` be able to accomplish this task, we need to compile the library with the required flags and also we need to have the reqeuired dependencies (as libcurl). Therefore to be able to read these files, without depending on these details, we need to generate a new local index and set the environment variables - :file:`REF_PATH` and :file:`REF_CACHE` - to the correct path:
# Create cache dir
mkdir -p /my/cache
# Construct the index
perl seq_cache_populate.pl -root /my/cache hg38.fa
# Now before running samtools or sider, we need to
# set the environment variables REF_PATH and REF_CACHE
export REF_PATH=/my/cache/%2s/%2s/%s:http://www.ebi.ac.uk/ena/cram
export REF_CACHE=/my/cache/%2s/%2s/%s
# So ...
sider ps -a annot.gff3.gz -o result file.cram
The script :file:`seq_cache_populate.pl` can be found in the :file:`samtools`, or at seq_cache_populate.pl.
For more information, see Samtools Worflow.
Now, let's do an interesting exercise, with real experimental data from the 1000 Genomes Project.
In order to run siderRETRO searching for retrocopies, we will download 2 whole-genome sequenced CRAM files, both aligned on the gencode's hg38 genome: NA12878 and NA12778.
At the beginning of a run, the files listed bellow must be at the same directory where the user is running sideRETRO or their correct paths must be supplied at the correspondent option. Files are:
- A GTF gene annotation file from gencode project (here :file:`gencode.v32.annotation.gtf`).
- A FASTA file with the gencode's Human reference genome, version 38 (here :file:`GRCh38_full_analysis_set_plus_decoy_hla.fa`).
- A custom perl script,
analyser.pl
, to do the final analysis over the VCF file and produce the TSV file in a tabular format. Theanalyser.pl
script can be downloaded :download:`here <data/analyser.pl>`.
Also, we will set the environment variables :file:`REF_PATH` and :file:`REF_CACHE`, as a requirement to work with CRAM files - more information at :ref:`Dealing with CRAM format<extern_cram>`.
See the complete command sequence bellow for the whole analysis:
# Do things inside a clean directory.
# Average time: irrelevant
$ mkdir -p sider_test
$ cd sider_test
# Download annotation from gencode
wget ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_32/gencode.v32.annotation.gtf.gz
# Download the reference genome from 1000 genomes
wget ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp/technical/reference/GRCh38_reference_genome/GRCh38_full_analysis_set_plus_decoy_hla.fa
# Make the CRAM index
# Create cache dir
mkdir -p cache
# create index
perl seq_cache_populate.pl -root cache GRCh38_full_analysis_set_plus_decoy_hla.fa
# Set environment variables
export REF_PATH=$PWD/cache/%2s/%2s/%s:http://www.ebi.ac.uk/ena/cram
export REF_CACHE=$PWD/cache/%2s/%2s/%s
# Create a download list (WGS.list) containing all files of interest.
# Average time: irrelevant
$ echo "ftp://ftp.sra.ebi.ac.uk/vol1/run/ERR323/ERR3239334/NA12878.final.cram" > WGS_download.list
$ echo "ftp://ftp.sra.ebi.ac.uk/vol1/run/ERR323/ERR3239484/NA12778.final.cram" >> WGS_download.list
# Download all files: NA12878 and NA12778.
# Average time: network dependent
$ wget -c -i WGS_download.list
# Create the list of BAM files.
# Average time: irrelevant
$ ls *.cram > WGS_genomes.list
# First sideRETRO step: process-sample
# Input file: WGS_genomes.list
# Output file: 1000_genomes.db
# Average time: 62m34.541
$ sider process-sample \
-i WGS_genomes.list \
-a gencode.v32.annotation.gtf.gz \
-p 1000_genomes \
-c 2000000 \
-Q 20 \
-F 0.9 \
-t 2
# Second sideRETRO step: merge-call
# Input file: 1000_genomes.db
# Output file: 1000_genomes.db (same file)
# Average time: 62m34.541
$ sider merge-call 1000_genomes.db \
-c 2000000 \
-x 1000000 \
-g 5 \
-I \
-t 2
# Second sideRETRO step: merge-call
# Input file: 1000_genomes.db
# Output file: 1000_genomes.vcf
# Average time: 62m34.541
$ sider make-vcf 1000_genomes.db \
-p 1000_genomes \
-r GRCh38_full_analysis_set_plus_decoy_hla.fa
# Some analysis over the final VCF file.
# Input file: 1000_genomes.vcf
# Output file: 1000_genomes.tsv
# Average time: 62m34.541
$ perl analyser.pl 1000_genomes.vcf > 1000_genomes.tsv
This was a simple but complete pipeline to obtain a final TSV file with all the relevant results in a tabular format ready to import in a R or Python script and plot some graphics.
Notwithstanding sideRETRO's native run, user can happily run it from a
Docker image just prepending Docker's directives to any example shown.
That is, supposing the user has Docker installed and has pulled the image
galantelab/sider:latest from DockerHub, he can just prepend
docker --rm -ti -v $(pwd):/home/sider -w /home/sider galantelab/sider
to the ordinary sider
command, like:
$ docker --rm -ti -v $(pwd):/home/sider -w /home/sider galantelab/sider \ sider ps \ -i my_files_list.txt \ -a annotation_file.gtf \ -o output_dir \ -p my_reads_database \ -l my_log_file.log \ -c 2000000 \ -Q 20 \ -F 0.9 \ -t 3