Hi Valerie,

thanks a lot for supporting legacy versions of the ensembl database /
variant_effect_predictor.pl script.

 I assume you're still using version 67 and have the data cached.


Yes, that's right. We use ensembl release 67 together with the
corresponding variant_effect_predictor.pl script version 2.5.


> How are you calling the script right now?


As a temporary fix, I am using the ensemblVEP method from ensemblVEP
version 1.1.3 (BioC svn revision r76970). I think this is the last version
that worked with ensembl release 67 for me.

I modified the default parameters in the VEPParam object by creating a
temporary "gVEPParam" class for use with our in-house ensembl release 67.
This object is passed to the ensemblVEP method with the default parameters
listed below. (Please note that our installation of
variant_effect_predictor.pl by default connects to our in-house database.)

Formal class 'gVEPParam' with 6 slots
  ..@ basic   :List of 5
  .. ..$ verbose    : logi FALSE
  .. ..$ quiet      : logi FALSE
  .. ..$ no_progress: logi TRUE
  .. ..$ config     : chr(0)
  .. ..$ everything : logi FALSE
  ..@ input   :List of 4
  .. ..$ species        : chr "homo_sapiens"
  .. ..$ format         : chr(0)
  .. ..$ output_file    : chr(0)
  .. ..$ force_overwrite: logi FALSE
  ..@ output  :List of 24
  .. ..$ terms      : chr "so"
  .. ..$ sift       : chr "b"
  .. ..$ polyphen   : chr "b"
  .. ..$ regulatory : logi FALSE
  .. ..$ cell_type  : chr(0)
  .. ..$ hgvs       : logi TRUE
  .. ..$ hgnc       : logi TRUE
  .. ..$ gene       : logi TRUE
  .. ..$ protein    : logi TRUE
  .. ..$ ccds       : logi TRUE
  .. ..$ canonical  : logi TRUE
  .. ..$ xref_refseq: logi FALSE
  .. ..$ numbers    : logi TRUE
  .. ..$ domains    : logi TRUE
  .. ..$ most_severe: logi FALSE
  .. ..$ summary    : logi FALSE
  .. ..$ per_gene   : logi FALSE
  .. ..$ convert    : chr(0)
  .. ..$ fields     : chr(0)
  .. ..$ vcf        : logi FALSE
  .. ..$ gvf        : logi FALSE
  .. ..$ original   : logi FALSE
  .. ..$ custom     : chr(0)
  .. ..$ plugin     : chr "GNECondel,/Plugins/config/Condel/config"
  ..@ filterqc:List of 17
  .. ..$ check_ref        : logi FALSE
  .. ..$ coding_only      : logi FALSE
  .. ..$ check_existing   : logi TRUE
  .. ..$ check_alleles    : logi FALSE
  .. ..$ check_svs        : logi FALSE
  .. ..$ individual       : chr(0)
  .. ..$ chr              : chr(0)
  .. ..$ no_intergenic    : logi FALSE
  .. ..$ filter_common    : logi FALSE
  .. ..$ check_frequency  : logi FALSE
  .. ..$ freq_pop         : chr(0)
  .. ..$ freq_freq        : logi FALSE
  .. ..$ freq_gt_lt       : chr(0)
  .. ..$ freq_filter      : chr(0)
  .. ..$ filter           : chr(0)
  .. ..$ failed           : logi FALSE
  .. ..$ allow_non_variant: logi FALSE
  ..@ database:List of 9
  .. ..$ database  : logi FALSE
  .. ..$ host      : chr "useastdb.ensembl.org"
  .. ..$ user      : chr(0)
  .. ..$ password  : chr(0)
  .. ..$ port      : num(0)
  .. ..$ genomes   : logi FALSE
  .. ..$ refseq    : logi FALSE
  .. ..$ db_version: num(0)
  .. ..$ registry  : chr(0)
  ..@ advanced:List of 4
  .. ..$ no_whole_genome: logi FALSE
  .. ..$ buffer_size    : num 5000
  .. ..$ compress       : chr(0)
  .. ..$ skip_db_check  : logi FALSE



> Do you use the --cache flag or --offline flag?


I am not using the --cache flag right now, because version 2.5 of the
variant_effect_predictor.pl script does not allow me to specify the Plugin
directory and the cache directory separately. (This was only introduced in
a later version of the perl script).

The --offline flag does not seem to be available in
variant_effect_predictor.pl  version 2.5, at least I cannot find it in the
listed arguments (provided below for reference).

version 2.5

Options
=======

--help                 Display this message and quit
--verbose              Display verbose output as the script runs [default:
off]
--quiet                Suppress status and warning messages [default: off]
--no_progress          Suppress progress bars [default: off]

--config               Load configuration from file. Any command line
options
                       specified overwrite those in the file [default: off]

--everything           Shortcut switch to turn on commonly used options.
See web
                       documentation for details [default: off]

-i | --input_file      Input file - if not specified, reads from STDIN.
Files
                       may be gzip compressed.
--format               Specify input file format - one of "ensembl",
"pileup",
                       "vcf", "hgvs", "id" or "guess" to try and work out
format.
-o | --output_file     Output file. Write to STDOUT by specifying -o STDOUT
- this
                       will force --quiet [default:
"variant_effect_output.txt"]
--force_overwrite      Force overwriting of output file [default: quit if
file
                       exists]
--original             Writes output as it was in input - must be used with
--filter
                       since no consequence data is added [default: off]
--vcf                  Write output as VCF [default: off]
--gvf                  Write output as GVF [default: off]
--fields [field list]  Define a custom output format by specifying a
comma-separated
                       list of field names. Field names normally present in
the
                       "Extra" field may also be specified, including those
added by
                       plugin modules. Can also be used to configure VCF
output
                       columns [default: off]

--species [species]    Species to use [default: "human"]

-t | --terms           Type of consequence terms to output - one of
"ensembl", "SO",
                       "NCBI" [default: ensembl]

--sift=[p|s|b]         Add SIFT [p]rediction, [s]core or [b]oth [default:
off]
--polyphen=[p|s|b]     Add PolyPhen [p]rediction, [s]core or [b]oth
[default: off]
--regulatory           Look for overlaps with regulatory regions. The
script can
                       also call if a variant falls in a high information
position
                       within a transcription factor binding site. Output
lines have
                       a Feature type of RegulatoryFeature or MotifFeature
                       [default: off]
--cell_type [types]    Report only regulatory regions that are found in the
given cell
                       type(s). Can be a single cell type or a
comma-separated list.
                       The functional type in each cell type is reported
under
                       CELL_TYPE in the output. To retrieve a list of cell
types, use
                       "--cell_type list" [default: off]
--custom [file list]   Add custom annotations from tabix-indexed files. See
                       documentation for full details [default: off]
--plugin [plugin_name] Use named plugin module [default: off]
--hgnc                 Add HGNC gene identifiers to output [default: off]
--hgvs                 Output HGVS identifiers (coding and protein).
Requires database
                       connection [default: off]
--ccds                 Output CCDS transcript identifiers [default: off]
--xref_refseq          Output aligned RefSeq mRNA identifier for
transcript. NB: the
                       RefSeq and Ensembl transcripts aligned in this way
MAY NOT, AND
                       FREQUENTLY WILL NOT, match exactly in sequence, exon
structure
                       and protein product [default: off]
--protein              Output Ensembl protein identifer [default: off]
--gene                 Force output of Ensembl gene identifer - disabled by
default
                       unless using --cache or --no_whole_genome [default:
off]
--canonical            Indicate if the transcript for this consequence is
the canonical
                       transcript for this gene [default: off]
--domains              Include details of any overlapping protein domains
[default: off]
--numbers              Include exon & intron numbers [default: off]

--no_intergenic        Excludes intergenic consequences from the output
[default: off]
--coding_only          Only return consequences that fall in the coding
region of
                       transcripts [default: off]
--most_severe          Ouptut only the most severe consequence per
variation.
                       Transcript-specific columns will be left blank.
[default: off]
--summary              Output only a comma-separated list of all
consequences per
                       variation. Transcript-specific columns will be left
blank.
                       [default: off]
--per_gene             Output only the most severe consequence per gene.
Where more
                       than one transcript has the same consequence, the
transcript
                       chosen is arbitrary. [default: off]
--check_ref            If specified, checks supplied reference allele
against stored
                       entry in Ensembl Core database [default: off]
--check_existing       If specified, checks for existing co-located
variations in the
                       Ensembl Variation database [default: off]
--failed [0|1]         Include (1) or exclude (0) variants that have been
flagged as
                       failed by Ensembl when checking for existing
variants.
                       [default: exclude]
--check_alleles        If specified, the alleles of existing co-located
variations
                       are compared to the input; an existing variation
will only
                       be reported if no novel allele is in the input
(strand is
                       accounted for) [default: off]
--check_svs            Report overlapping structural variants [default: off]

--filter [filters]     Filter output by consequence type. Use this to
output only
                       variants that have at least one consequence type
matching the
                       filter. Multiple filters can be used separated by
",". By
                       combining this with --original it is possible to run
the VEP
                       iteratively to progressively filter a set of
variants. See
                       documentation for full details [default: off]

--check_frequency      Turns on frequency filtering. Use this to include or
exclude
                       variants based on the frequency of co-located
existing
                       variants in the Ensembl Variation database. You must
also
                       specify all of the following --freq flags [default:
off]
--freq_pop [pop]       Name of the population to use e.g. hapmap_ceu for
CEU HapMap,
                       1kg_yri for YRI 1000 genomes. See documentation for
more
                       details
--freq_freq [freq]     Frequency to use in filter. Must be a number between
0 and 0.5
--freq_gt_lt [gt|lt]   Specify whether the frequency should be greater than
(gt) or
                       less than (lt) --freq_freq
--freq_filter          Specify whether variants that pass the above should
be included
  [exclude|include]    or excluded from analysis

--individual [id]      Consider only alternate alleles present in the
genotypes of the
                       specified individual(s). May be a single individual,
a comma-
                       separated list or "all" to assess all individuals
separately.
                       Each individual and variant combination is given on
a separate
                       line of output. Only works with VCF files containing
individual
                       genotype data; individual IDs are taken from column
headers.
--allow_non_variant    Prints out non-variant lines when using VCF input

--chr [list]           Select a subset of chromosomes to analyse from your
file. Any
                       data not on this chromosome in the input will be
skipped. The
                       list can be comma separated, with "-" characters
representing
                       a range e.g. 1-5,8,15,X [default: off]
--gp                   If specified, tries to read GRCh37 position from GP
field in the
                       INFO column of a VCF file. Only applies when VCF is
the input
                       format and human is the species [default: off]

--convert              Convert the input file to the output format
specified.
  [ensembl|vcf|pileup] Converted output is written to the file specified in
                       --output_file. No consequence calculation is carried
out when
                       doing file conversion. [default: off]

--refseq               Use the otherfeatures database to retrieve
transcripts - this
                       database contains RefSeq transcripts (as well as
CCDS and
                       Ensembl EST alignments) [default: off]
--host                 Manually define database host [default: "
ensembldb.ensembl.org"]
-u | --user            Database username [default: "anonymous"]
--port                 Database port [default: 5306]
--password             Database password [default: no password]
--genomes              Sets DB connection params for Ensembl Genomes
[default: off]
--registry             Registry file to use defines DB connections
[default: off]
                       Defining a registry file overrides above connection
settings.
--db_version=[number]  Force script to load DBs from a specific Ensembl
version. Not
                       advised due to likely incompatibilities between API
and DB

--no_whole_genome      Run in old-style, non-whole genome mode [default:
off]
--buffer_size          Sets the number of variants sent in each batch
[default: 5000]
                       Increasing buffer size can retrieve results more
quickly
                       but requires more memory. Only applies to whole
genome mode.

--cache                Enables read-only use of cache [default: off]
--dir [directory]      Specify the base cache directory to use [default:
"$HOME/.vep/"]
--write_cache          Enable writing to cache [default: off]
--build [all|list]     Build a complete cache for the selected species.
Build for all
                       chromosomes with --build all, or a list of
chromosomes (see
                       --chr). DO NOT USE WHEN CONNECTED TO PUBLIC DB
SERVERS AS THIS
                       VIOLATES OUR FAIR USAGE POLICY [default: off]

--compress             Specify utility to decompress cache files - may be
"gzcat" or
                       "gzip -dc" Only use if default does not work
[default: zcat]

--skip_db_check        ADVANCED! Force the script to use a cache built from
a different
                       database than specified with --host. Only use this
if you are
                       sure the hosts are compatible (e.g.
ensembldb.ensembl.org and
                       useastdb.ensembl.org) [default: off]
--cache_region_size    ADVANCED! The size in base-pairs of the region
covered by one
                       file in the cache. [default: 1MB]



> Also, please remind me of (point me to) the plug-in you're using so I can
> test that.
>

We are using a single plugin that returns the Condel scores. The *Condel
plugin* can be found on github here:
https://github.com/ensembl-variation/VEP_plugins

Again, thanks a lot for your support. Please let me know if there is
anything I can do to help, e.g. with testing the package.

Best,
Thomas

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