-
Notifications
You must be signed in to change notification settings - Fork 201
Primer Filter
vg primers can be used to filter PRC primers based on properties of the pangenome such as whether there are variations in the primers and the possible lengths of the PRC product.
vg primers takes pangenome indexes and the output of primer3 as input and outputs a .tsv file of the input primers and properties from the pangenome.
The input to vg primers is the output of the command line version of primer3.
primer3 requires a config file formatted like:
SEQUENCE_ID=CHM13#0#chr17|BRCA1P1|exon_1|44026826
SEQUENCE_TEMPLATE=CATGT...
PRIMER_NUM_RETURN=10
PRIMER_TASK=generic
PRIMER_PICK_LEFT_PRIMER=1
PRIMER_PICK_INTERNAL_OLIGO=0
PRIMER_PICK_RIGHT_PRIMER=1
PRIMER_OPT_SIZE=20
PRIMER_MIN_SIZE=18
PRIMER_MAX_SIZE=22
PRIMER_PRODUCT_SIZE_RANGE=75-150
PRIMER_EXPLAIN_FLAG=1
=
The SEQUENCE_ID field must be formatted correctly for vg primers to find the correct location of the primers in the pangenome.
There are four fields in SEQUENCE_ID separated by |.
They are the reference path name in the graph, the name of the gene, the exon or intron, and the offset of the sequence in the path.
The names of the reference paths in the graph can be found with vg paths -L -R.
The SEQUENCE_TEMPLATE is the nucleotide sequence the primers are found from.
vg primers requires the following indexes of the pangenome:
- the xg index created with
vg index -x - the distance index created with
vg index -j - the r-index created with
vg gbwt -r - the gbz created with
vg gbwt
If the SEQUENCE_ID field is not formatted correctly and the reference coordinates cannot be found, then vg primers will try to find the reference coordinates by mapping the template sequence with vg giraffe then projecting onto the reference with vg surject.
This process is not recommended because it is not guaranteed that a good alignment will be found.
If the sequence needs to be mapped, then vg primers additionally requires the following files:
- the minimizer index created with
vg minimizer. We recommend that this is run with-k 31 -w 50 -Wand-zto create the zipcode file - the zipcode file, also created with vg minimizer
vg primers outputs a tsv file with the following fields for each primer:
| field | definition | description |
|---|---|---|
| chrom | chromosome | reference path name, the first field in SEQUENCE_ID
|
| tplfeat | template feature | the second and third fields in SEQUENCE_ID
|
| tplpos | template position | offset along the reference path, the fourth field in SEQUENCE_ID
|
| lpseq | left primer sequence | the nucleotide sequence of the left primer |
| rpseq | right primer sequence | the nucleotide sequence of the right primer |
| lppostpl | left primer position template | the offset of the left primer in the template sequence |
| rppostmp | right primer position template | the offset of the right primer in the template sequence |
| lpposchrom | left primer position chromosome | the offset of the left primer in the reference path |
| rpposchrom | right primer position chromosome | the offset of the right primer in the reference path |
| pnid | left primer mapped node ids | the node ids that the left primer overlaps in the graph |
| rpnid | right primer mapped node ids | the node ids that the right primer overlaps in the graph |
| lplen | left primer length | the length in nucleotides of the left primer |
| rplen | right primer length | the length in nucleotides of the right primer |
| linsize | linear product size | the length of the product in the linear genome (including primer lengths) |
| minsize | minimum product size | the minimum length of the product according to the pangenome |
| maxsize | maximum product size | the maximum length of the product according to the pangenome |
| varlevel | variation level | a measure of variation in the primers (the number of unique haplotypes / the total number of haplotypes) |