For Users: CLI

The CLI version deploys a command called primertool.

Running modes

Full mode

$ primertool full -h

In this mode, primertool accepts target regions or target sequences (called query) defined by users, designs primers from these regions, and searches user defined databases (called templates) to defined primer specificities.

Design mode

$ primertool design -h

In this mode, primertool just designs primers and doesn't conduct specificity check. This is usually used in NGS target sequencing panels where primer specificity is not so necessary as in legacy PCR.

Checking mode

$ primertool check -h

In this mode, primertool accepts a set of user defined primer sequences (called query) and check their specificity against certain databases (called templates).

Input (REQUIRED):

  query                 query file. (STDIN is acceptable)
  templates             template file in FASTA format. Allowing multiple files
                        (separated by comma), where the first one is used to
                        design primers and/or order the primer specificity

Overall Setttings

  --primer-num-retain PRIMER_NUM_RETAIN
                        The maximum number of primers to retain in each site
                        in the final report. (default: 10)
  --check-multiplex     Checking dimers between primers in different sites,
                        which is useful in multiplex PCR. (default: False)
  --Tm-diff TM_DIFF     The mininum difference of melting temperature (℃)
                        suggested to produce off-target amplicon or primer
                        dimers. Suggest >10 (default: 20)
  -p CPU, --cpu CPU     Used CPU number. (default: 2)
  -o OUT, --out OUT     Output primers in JSON format. default: {query}.json
                        (default: None)
  -t TSV, --tsv TSV     Output primers in TSV format. default: {query}.tsv
                        (default: None)

Mode specific settings

Full mode and Design mode

  --type {SEQUENCE_TARGET,SEQUENCE_INCLUDED_REGION,FORCE_END}
                        designing primer types (default: SEQUENCE_TARGET)
  --pick-oligo          Pick internal Oligos (Probes) for qRT-PCR (default:
                        False)
  --product-size-min PRODUCT_SIZE_MIN
                        Lower limit of the product amplicon size range (bp).
                        (default: 70)
  --product-size-max PRODUCT_SIZE_MAX
                        Upper limit of the product amplicon size range (bp).
                        (default: 1000)
  --Tm-opt TM_OPT       Optmized melting temperature for primers (℃).
                        (default: 60)
  --primer-num-return PRIMER_NUM_RETURN
                        The maximum number of primers to return in Primer3
                        designing results. (default: 30)
  --junction            Primer pair must be separated by at least one intron
                        on the corresponding genomic DNA; or primers must span
                        an exon-exon junction. Junction data in JSON format
                        should be prepared by the command-line primertool-
                        junctions (default: False)

As to the details of the --type option, please refer to the legacy PrimerServer wiki. Generally speaking:

• SEQUENCE_TARGET: the default value, used in target sequencing (SSR, SNPs, and so on)
• SEQUENCE_INCLUDED_REGION: used in qPCR quantification
• FORCE_END: used in SNP genotyping.

Full mode and Check mode

  -3, --use-3-end       If turned on, primer pairs having at least one
                        mismatch at the 3 end position with templates would
                        not be considered to produce off-target amplicon, even
                        if their melting temperatures are high. Turn on this
                        would find more candidate primers, but might also have
                        more false positives (default: False)
  --checking-size-min CHECKING_SIZE_MIN
                        Lower limit of the checking amplicon size range (bp).
                        (default: 50)
  --checking-size-max CHECKING_SIZE_MAX
                        Upper limit of the checking amplicon size range (bp).
                        (default: 2000)
  --amplicon-num-max AMPLICON_NUM_MAX
                        The maximum number of amplicons for checking.
                        (default: 10)
  -a, --report-amplicon-seqs
                        Get amplicon seqs (might be slow) (default: False)
  --isoform             Allow primers targeting on alternative isoforms and
                        still regard them as specific ones. Isoform data in
                        JSON format should be prepared by the command-line
                        primertool-isoforms (default: False)