Major varVAMP update to version 1.2

These changes are credited to @wm75 (#39).

CHANGELOG

(1) Changes around BLAST functionality:

• Off-target amplicons are now reported as intended in the logs
• Off-target amplicons are now always considered last for the final scheme (no penalty is used, but the fact that they had BLAST matches gets recorded)
• The BLAST_PENALTY config option is no longer needed and has been removed
• Added a new off_target_amplicons column to the qPCR design, the qPCR primer and the (single/tiled) primer tsv outputs to indicate which final amplicons had BLAST hits

(2) General reporting changes:

• Amplicon numbering now proceeds from 5' to 3' even across pools for the tiled mode and from lowest penalty to highest for the other modes (previously additional BLAST penalties weren't considered during penalty-sorting).
• In the primer bed file output in tiled mode, primers are now ordered according to the amplicon number without taking the pool into account
• In the primer bed file output in qPCR mode, oligos from the same set are now ordered LEFT, PROBE, RIGHT, i.e. by position on the reference
• The per-base mismatch plot now uses final primer names as panel titles
• The amplicon bed file, in all modes, is now formatted as proper six-column bed

(3) Algorithmic fixes and enhancements:

• The internal representation of amplicon schemes has been unified/simplified across the different modes and steps of the analysis
• Search for final non-overlapping amplicons in single mode and for non-overlapping amplicons passing deltaG in qpcr mode has been optimized and is now significantly faster
• Some off-by-one errors in internal primer and amplicon interval calculations have been fixed - generally more primers are now found and considered

Full Changelog: v.1.1.3...v.1.2.0

Proper v.1.1.3 release with correct varVAMP version.

Minor bug fix

Fixed bug in the BLAST - qPCR module interface.

Some minor fixes:

• END_OVERLAP is now getting logged and error is reported if set to negative values.
• fixed a bug that caused a crash in the qpcr mode if -a = 0 and -pa not set. Now it sets -pa to 0.

HOTFIX

Fixes:

• fixed error with the qPCR mode in combination with a blast db that occured due to the renaming of the primers
• harmonized primer naming in the code at multiple places for consistency - now all primers are named based on their direction or function as: LEFT RIGHT or PROBE

NEW:

• introduced a config class that enables the user to provide a custom varVAMP config file. Instead of changing the config.py file, the user can can prepend VARVAMP_CONFIG=<path/to/custom_config> to any regular varvamp command line without any changes to the code basis.

FIXES:

• consistent primer naming over all modes
• blast is now called by a sub-process as biopython's blast module will be removed in the future.
• updated the documentation
• fixed the --threads option in combination with -db

Large thanks to @wm75 for these improvements!

NEW
changed SANGER mode to SINGLE mode. Better naming to what this modus actually does. Design primers for single amplicons.

NEW

• added fasta output for primers
• new workflow.png in the "How varVAMP works" section
• renamed n_threads argument to threads

FIXES

• renamed scores to penalties at several pos in the code to avoid confusion.
• code reformatting and clearer documentation
• more intuitive way of how polyX and polyXY are counted (prior three repeat was equal to the Primer setting MAX_POLYX = 3, but meant that stretches of e.g. 4 As were tolerated). Now the counting was adjusted so that MAX_POLYX = 4 is equal to the prior MAX_POLYX = 3.
• renamed primer names in a more intuitive manner. The old primer names are now given as alternate names in the tsv.

FIXES:

Fixed entropy calculation for the final plot as the normalization was not correct in prior versions. Entropy is now correctly normalized (prior there was some miscalculation with the log base).

Update to v.0.9.4

No new features but major changes to how many primers are found and quite a lot of bug fixes that appeared during stress-testing.

NEW:

• overhauled best primer search (now only excludes primers that overlap with the middle third of a high scoring primer) increasing the amount of primers that are found roughly 2.5 times. Mostly useful for very short amplicons (<400 bp) where the optimal primers were too widely spaced. Now primers adjacent or minimally overlapping are found.
• massively increased kmer digestion runtime (set instead of list)
• amplicon penalty is increased for amplicons longer than the optimum (e^(fold length) instead of fold length) biasing the scheme towards the shorter more optimal amplicons
• updated docs
• tsv files start positions are now 1 based instead of 0 based to avoid confusion
• overhauled dimer search in scheme. Previously could not exit while loop in some cases. Now it tries to solve primer dimers once and reports any dimer pairs that after the initial solve appear (not perfect but should catch most primer dimers)

TWEAKS to get slightly more primers:

• increased MAX_BASE_PENALTY to 10
• decreased primer min temp by 1°C

BUGFIXES:

• fixed bug that caused the alignment to be trimmed massively if there was a deletion of the length 1
• fixed warnings caused by non-closed figures
• fixed unintended random sorting of primers with the same score
• fixed a bug in dimer reporting
• BLAST installation check is now performed for all modi