conda install longshot

conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge

sudo apt-get install cargo zlib1g-dev xz-utils \
         libclang-dev clang cmake build-essential curl git  # install dependencies 
git clone https://github.com/pjedge/longshot          # clone the Longshot repository
cd longshot                                           # change directory
cargo install --path .                                # install Longshot
export PATH=$PATH:/home/$USER/.cargo/bin              # add cargo binaries to path

$ longshot [FLAGS] [OPTIONS] --bam <BAM/CRAM> --ref <FASTA> --out <VCF>

longshot --bam example_data/pacbio_reads_30x.bam --ref example_data/genome.fa --out example_data/longshot_output.vcf

$ longshot --help

Longshot: variant caller (SNVs) for long-read sequencing data 

USAGE:
    longshot [FLAGS] [OPTIONS] --bam <BAM/CRAM> --ref <FASTA> --out <VCF>

FLAGS:
    -A, --auto_max_cov        Automatically calculate mean coverage for region and set max coverage to mean_coverage +
                              5*sqrt(mean_coverage). (SLOWER)
    -S, --stable_alignment    Use numerically-stable (logspace) pair HMM forward algorithm. Is significantly slower but
                              may be more accurate. Tests have shown this not to be necessary for highly error prone
                              reads (PacBio CLR).
    -F, --force_overwrite     If output files (VCF or variant debug directory) exist, delete and overwrite them.
    -x, --max_alignment       Use max scoring alignment algorithm rather than pair HMM forward algorithm.
    -n, --no_haps             Don't call HapCUT2 to phase variants.
        --output-ref          print reference genotypes (non-variant), use this option only in combination with -v
                              option.
    -h, --help                Prints help information
    -V, --version             Prints version information

OPTIONS:
    -b, --bam <BAM>                            sorted, indexed BAM file with error-prone reads (CRAM files also supported)
    -f, --ref <FASTA>                          indexed FASTA reference that BAM file is aligned to
    -o, --out <VCF>                            output VCF file with called variants.
    -r, --region <string>                      Region in format <chrom> or <chrom:start-stop> in which to call variants
                                               (1-based, inclusive).
    -v, --potential_variants <VCF>             Genotype and phase the variants in this VCF instead of using pileup
                                               method to find variants. NOTES: VCF must be gzipped and tabix indexed or
                                               contain contig information. Use with caution because excessive false
                                               potential variants can lead to inaccurate results. Every variant is used
                                               and only the allele fields are considered -- Genotypes, filters,
                                               qualities etc are ignored. Indel variants will be genotyped but not
                                               phased. Structural variants (length > 50 bp) are currently not supported.
    -O, --out_bam <BAM>                        Write new bam file with haplotype tags (HP:i:1 and HP:i:2) for reads
                                               assigned to each haplotype, any existing HP and PS tags are removed
    -c, --min_cov <int>                        Minimum coverage (of reads passing filters) to consider position as a
                                               potential SNV. [default: 6]
    -C, --max_cov <int>                        Maximum coverage (of reads passing filters) to consider position as a
                                               potential SNV. [default: 8000]
    -q, --min_mapq <int>                       Minimum mapping quality to use a read. [default: 20]
    -a, --min_allele_qual <float>              Minimum estimated quality (Phred-scaled) of allele observation on read to
                                               use for genotyping/haplotyping. [default: 7.0]
    -y, --hap_assignment_qual <float>          Minimum quality (Phred-scaled) of read->haplotype assignment (for read
                                               separation). [default: 20.0]
    -Q, --potential_snv_cutoff <float>         Consider a site as a potential SNV if the original PHRED-scaled QUAL
                                               score for 0/0 genotype is below this amount (a larger value considers
                                               more potential SNV sites). [default: 20.0]
    -e, --min_alt_count <int>                  Require a potential SNV to have at least this many alternate allele
                                               observations. [default: 3]
    -E, --min_alt_frac <float>                 Require a potential SNV to have at least this fraction of alternate
                                               allele observations. [default: 0.125]
    -L, --hap_converge_delta <float>           Terminate the haplotype/genotype iteration when the relative change in
                                               log-likelihood falls below this amount. Setting a larger value results in
                                               faster termination but potentially less accurate results. [default:
                                               0.0001]
    -l, --anchor_length <int>                  Length of indel-free anchor sequence on the left and right side of read
                                               realignment window. [default: 6]
    -m, --max_snvs <int>                       Cut off variant clusters after this many variants. 2^m haplotypes must be
                                               aligned against per read for a variant cluster of size m. [default: 3]
    -W, --max_window <int>                     Maximum "padding" bases on either side of variant realignment window
                                               [default: 50]
    -I, --max_cigar_indel <int>                Throw away a read-variant during allelotyping if there is a CIGAR indel
                                               (I/D/N) longer than this amount in its window. [default: 20]
    -B, --band_width <Band width>              Minimum width of alignment band. Band will increase in size if sequences
                                               are different lengths. [default: 20]
    -D, --density_params <string>              Parameters to flag a variant as part of a "dense cluster". Format
                                               <n>:<l>:<gq>. If there are at least n variants within l base pairs with
                                               genotype quality >=gq, then these variants are flagged as "dn" [default:
                                               10:500:50]
    -s, --sample_id <string>                   Specify a sample ID to write to the output VCF [default: SAMPLE]
        --hom_snv_rate <float>                 Specify the homozygous SNV Rate for genotype prior estimation [default:
                                               0.0005]
        --het_snv_rate <float>                 Specify the heterozygous SNV Rate for genotype prior estimation [default:
                                               0.001]
        --ts_tv_ratio <float>                  Specify the transition/transversion rate for genotype grior estimation
                                               [default: 0.5]
    -P, --strand_bias_pvalue_cutoff <float>    Remove a variant if the allele observations are biased toward one strand
                                               (forward or reverse) according to Fisher's exact test. Use this cutoff
                                               for the two-tailed P-value. [default: 0.01]
    -d, --variant_debug_dir <path>             write out current information about variants at each step of algorithm to
                                               files in this directory

longshot --bam pacbio.bam --ref ref.fa --out output.vcf

longshot -A -r chr1 --bam pacbio.bam --ref ref.fa --out output.vcf

longshot -r chr1:1000000-1500000 -y 30 -O reads.bam --bam pacbio.bam --ref ref.fa --out output.vcf

rustc --version

rustup update

error: linking with `cc` failed: exit code: 1
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= note: Non-UTF-8 output: /usr/bin/ld: /home/pedge/temp/longshot/target/release/build/longshot-347f3774e75b380c/out/libhapcut2.a(common.o)(.text.fprintf_time+0x81): unresolvable H\x89\\$\xe8H\x89l$\xf0H\x89\xf3L\x89d$\xf8H\x83\xec\x18H\x8bG\x10H\x89\xfdI\x89\xd4H\x89\xd6H\x8b;\xffPxH\x8bE\x10I\x8dt$\x08H\x8b{\x08\xffPxH\x8bE\x10H\x8b{\x10I\x8dt$\x10H\x8b\x1c$H\x8bl$\x08L\x8bd$\x10H\x8b@xH\x83\xc4\x18\xff\xe0f\x90H\x89\\$\xe8H\x89l$\xf0H\x89\xfbL\x89d$\xf8H\x83\xec\x18H\x8bG\x10I\x89\xd4H\x89\xf5H\x89\xf7\xffPhI\x89\x04$H\x8bC\x10H\x8d}\x08\xffPhH\x8b\x1c$I\x89D$\x08H\x8bl$\x08L\x8bd$\x10H\x83\xc4\x18\xc3\x0f\x1f relocation against symbol `time@@GLIBC_2.2.5\'\n/usr/bin/ld: BFD version 2.20.51.0.2-5.42.el6 20100205 internal error, aborting at reloc.c line 443 in bfd_get_reloc_size\n\n/usr/bin/ld: Please report this bug.\n\ncollect2: ld returned 1 exit status\n
...
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...

rustc -vV

mkdir .cargo
nano .cargo/config

[target.<target-name>]
linker = "</path/to/linker>"

[target.x86_64-unknown-linux-gnu]
linker = "/opt/gnu/gcc/bin/gcc"

cargo clean
cargo build --release