### Minimum Read Support Criteria for Variant Calling in pbsv

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

Details the minimum read support requirements across all samples and individual samples for a variant to be considered. This includes total reads, reads per sample, and percentage of reads per sample. Specific thresholds are provided for BNDs and reads per strand.

```bash
* supported by at least `-A,--call-min-reads-all-samples [3]` reads total across samples,
* supported by at least `-O,--call-min-reads-one-samples [3]` in a sample,
* supported by at least `-S,--call-min-read-per-one-sample [1]` percent of reads in a sample,
* supported by at least `-B,--call-min-bnd-reads-all-samples [2]` reads total across samples for BNDs,
* supported by at least `-P,--call-min-reads-per-strand-all-samples [20]` reads per strand total across samples,
* assigned a non-reference genotype in at least one sample; a sample is assigned a non-reference genotype for a variant if at least `--gt-min-reads [1]` reads support the variant.
```

--------------------------------

### Discover Structural Variation Signatures

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

This command discovers signatures of structural variation from aligned BAM files. It outputs a compressed file containing these signatures, which is used for variant calling. Indexing with `tabix` is recommended for efficient random access.

```sh
pbsv discover ref.movie1.bam ref.sample1.svsig.gz
pbsv discover ref.movie2.bam ref.sample2.svsig.gz

# optionally index svsig.gz to allow random access via `pbsv call -r`
tabix -c '#' -s 3 -b 4 -e 4 ref.sample1.svsig.gz
tabix -c '#' -s 3 -b 4 -e 4 ref.sample2.svsig.gz
```

--------------------------------

### Align PacBio Reads using pbmm2 (CCS FASTQ)

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

This command aligns PacBio CCS FASTQ files to a reference genome using `pbmm2`. It sorts the output, uses a preset for CCS reads, assigns a sample name, and specifies the read group header. The output is a sorted BAM file.

```sh
pbmm2 align ref.fa movie1.Q20.fastq ref.movie1.bam --sort --preset CCS --sample sample1 --rg '@RG\tID:movie1'
```

--------------------------------

### Maximum Insertion Length Configuration in pbsv

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

Sets an upper limit on the length of insertions that pbsv will consider. Insertions exceeding this threshold are ignored. Be aware that increasing this limit can significantly increase memory consumption.

```bash
--max-ins-length   Ignore insertions with length > N bp. ["15K"]
```

--------------------------------

### Max Skip Split Configuration for Inversions and Translocations in pbsv

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

Defines the maximum permitted reference overlap between consecutive alignments for inversion detection and query skip for translocation detection. This parameter is configured in `pbsv discover` and `pbsv call`.

```bash
-k,--max-skip-split   Ignore alignment pairs separated by > N bp of a read or reference. ["100"]
```

--------------------------------

### Parallel SV Discovery per Chromosome

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

This script generates separate `.svsig.gz` files for each chromosome of a reference genome, enabling parallel processing. It iterates through the sequence dictionary in the BAM header and runs `pbsv discover` for each region.

```sh
for i in $(samtools view -H hg38.movie1.bam | grep '^@SQ' | cut -f2 | cut -d':' -f2); do
    pbsv discover --region $i hg38.movie1.bam hg38.sample1.$i.svsig.gz
done
```

--------------------------------

### Align PacBio Reads using pbmm2 (Subreads BAM)

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

This command aligns PacBio subreads BAM files to a reference genome using `pbmm2`. It sorts the output, applies a median filter, and assigns a sample name. The output is a sorted BAM file suitable for downstream analysis.

```sh
pbmm2 align ref.fa movie1.subreads.bam ref.movie1.bam --sort --median-filter --sample sample1
```

--------------------------------

### Maximum Duplication Length Configuration in pbsv

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

Specifies the maximum length for duplications that pbsv will report. Duplications exceeding this configurable limit are ignored. This parameter is set using `pbsv call`.

```bash
--max-dup-length   Ignore duplications with length > N bp. ["1M"]
```

--------------------------------

### SV Signature Cluster Options for Insertions in pbsv

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

Configures the clustering of structural variant signatures for insertions, specifically incorporating a minimum basepair identity threshold. This option is crucial for accurately grouping insertion signatures.

```bash
SV Signature Cluster Options:
  --cluster-min-basepair-perc-id   Do not cluster signatures with basepair identity < P%. [10]
```

--------------------------------

### Align PacBio Reads using pbmm2 (CCS BAM)

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

This command aligns PacBio CCS BAM files to a reference genome using `pbmm2`. It sorts the output, uses a preset optimized for CCS reads, and assigns a sample name. The output is a sorted BAM file.

```sh
pbmm2 align ref.fa movie1.ccs.bam ref.movie1.bam --sort --preset CCS --sample sample1
```

--------------------------------

### Call Structural Variants (Joint Calling)

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

This command calls structural variants and assigns genotypes from one or more `.svsig.gz` files. It can process multiple samples jointly, outputting all variant calls in a single VCF file. Add `--ccs` if input is from CCS reads.

```sh
pbsv call ref.fa ref.sample1.svsig.gz ref.sample2.svsig.gz ref.var.vcf
```

--------------------------------

### Parallel SV Calling

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

This command calls structural variants in parallel across multiple chromosomes. It takes the reference genome and all chromosome-specific `.svsig.gz` files as input, using the `-j` option to specify the number of threads for joint calling.

```sh
# -j is number of threads
pbsv call -j 8 hg38.fa hg38.sample1.*.svsig.gz hg38.sample1.vcf
```

--------------------------------

### SV Signature Cluster Options for Deletions in pbsv

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

Configures how structural variant signatures are clustered based on length percentage difference and reference position difference. These options are used during the `pbsv call` command for deletion detection. Default values are provided.

```bash
SV Signature Cluster Options:
  --cluster-max-length-perc-diff   Do not cluster signatures with difference in length > P%. [25]
  --cluster-max-ref-pos-diff       Do not cluster signatures > N bp apart in reference. [200]
```

--------------------------------

### Relaxed Thresholds for CCS Input in pbsv

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

Presents the relaxed calling thresholds applicable when using the `--ccs` mode in `pbsv call`. These adjusted parameters are intended for Circular Consensus Sequence (CCS) input data, potentially allowing for more sensitive variant detection.

```bash
For CCS input, using the `--ccs` mode in `pbsv call`, thresholds are relaxed to `-A 3 -B 2 -O 3 -S 0 -P 10`.
```

--------------------------------

### VCF Filtering Criteria in pbsv

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

Outlines the various criteria used to populate the VCF filter column. Variants are filtered based on proximity to reference gaps, involvement of decoy sequences, distance from contig ends, strand evidence, and whether duplications are fully spanned. Configuration parameters for some filters are included.

```bash
1) **PASS**
2) **NearReferenceGap**: variant is near (< `--filter-near-reference-gap [1K]`) from a gap (run of >= 50 Ns in the reference assembly)
3) **Decoy**: variant involves a decoy sequence, where the chromosome name contains `decoy`, `hs37d5`, or `hs38d1`
4) **NearContigEnd**: variant is near (< `--filter-near-contig-end [1K]`) from a contig end
5) **InsufficientStrandEvidence**: variant is not supported by at least (`--call-min-reads-per-strand-all-samples [1]`) reads in forward and reverse orientation
6) **NotFullySpanned**: duplication variant does not have any fully spanning reads
```

--------------------------------

### Consensus Generation Options for Deletions in pbsv

Source: https://github.com/pacificbiosciences/pbsv/blob/master/README.md

Limits the number of reads used for variant consensus generation during the deletion calling process. This helps manage computational resources and potential noise. A default limit is specified.

```bash
Consensus Options:
  -x,--max-consensus-coverage      Limit to N reads for variant consensus. [20]
```

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