--- name: "sider" section: "1" platform: "linux" package: "sideretro" version: "1.1.6-2" release: "Debian 13 (Trixie)" platforms: ["linux"] url: "https://new.carta.tech/sider" --- # sider(1) sideRETRO Documentation ## NAME sider - sideRETRO Documentation ## GENERAL SYNTAX **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 [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 *A Practical Workflow* and *Running with Docker* 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. ## COMMAND PROCESS-SAMPLE 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 *A Practical Workflow* section. ## COMMAND MERGE-CALL 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 *A Practical Workflow* section. ## COMMAND MAKE-VCF 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. ## DEALING WITH CRAM FORMAT 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 ### Local 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.cram** can be processed with **sider**, as long as we don't change the reference genome and its index (**.fa.fai**) path. If so, we need to set the environment variables **REF\_PATH** and **REF\_CACHE**, see *External alignment*. ### External alignment 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 **.cram**, and the **htslib** library - used by **sider** and **samtools** - is able to download the index from the *CRAM Reference Registry*. However, in order to **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 - **REF\_PATH** and **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 **seq\_cache\_populate.pl** can be found in the **samtools**, or at *seq\_cache\_populate.pl*. For more information, see *Samtools Worflow*. ## A PRACTICAL WORKFLOW Now, let's do an interesting exercise, with real experimental data from the *1000 Genomes Project*. (Warning: This example requires 16GB of RAM) 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 below 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: - 1\. A GTF gene annotation file from gencode project (here **gencode.v32.annotation.gtf**). - 2\. A FASTA file with the gencode's Human reference genome, version 38 (here **GRCh38\_full\_analysis\_set\_plus\_decoy\_hla.fa**). - 3\. A custom perl script, **seq\_cache\_populate.pl**, to construct a new local index . The **seq\_cache\_populate.pl** script can be found in *seq\_cache\_populate.pl*. - 4\. A custom perl script, **analyser.pl**, to do the final analysis over the VCF file and produce the TSV file in a tabular format. The **analyser.pl** script can be downloaded **here**. Also, we will set the environment variables **REF\_PATH** and **REF\_CACHE**, as a requirement to work with CRAM files - more information at *Dealing with CRAM format*. See the complete command sequence below for the whole analysis. Tip: Copy and paste line by line in your terminal. Tip 2: If you are running line by line in your terminal don't paste the "$" character. It is already in your terminal. ``` # 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. ## RUNNING WITH DOCKER 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 ``` ## AUTHOR Thiago L. A. Miller ## COPYRIGHT 2020, The sideRETRO Team