This is a bug fix release, it should make no difference in AMRFinderPlus results

AMRFinderPlus 4.2.7 contains a fix for issue #180, a bug that caused AMRFinderPlus to crash when an HMM hit to a protein is not detected by any blast alignments. This appears to be a rare situation, at least in Pathogen Detection data. We saw one incident in ~200,000 annotated assemblies, but possibly it is more common in other datasets.

It also includes some minor improvements in curl code and testing.

This is a bug fix release (#174).

Version 4.2.4 run with a downloaded database with the --organism Klebsiella_pneumoniae option failed with released version 4.2.4 because the automatic database download missed an essential file.

Please install version 4.2.5 and run amrfinder --force_update to re-download the complete AMRFinderPlus database.

AMRFinderPlus version 4.2.4 has a few significant changes. The most significant is we added the ability for AMRFinderPlus to identify putatively function disrupting mutations in genes that lead to resistance when disrupted (frame shifts and stop codons) . Calls for these genes will have a subtype of POINT_DISRUPT.

As part of this new functionality we slightly revised our standard point mutation naming system to handle the sometimes complex mutations identified by the new POINT_DISRUPT module and to bring them closer to the HGVS standard. "STOP" became "Ter" and Frame shifts will identify the length of sequence before the stop codon E.g., cirA_Y253CfsTer5. See https://github.com/ncbi/amr/wiki/Point-mutation-nomenclature for details.

Another change was made to better handle an edge case we encountered. The rules designed to get correct call on partial fusion genes were changed where the previous versions excluded extended proteins with > 20-aa that don't align. Version 4.2.4 allows a fusion gene call to override the smaller individual subunit call if 1 or more amino-acids align to the fusion gene than the subunit gene.

Concurrent with making these changes we also significantly refactored the code to interpret blast output for both AMRFinderPlus and StxTyper and made for slightly improved alignments. These changes can change match statistics (e.g., % Identity and % Coverage) for the following reasons:

• AMRFinderPlus improved translated BLAST alignments when the final amino-acid is a mismatch, this sometimes causes AMRFinderPlus version 4.2.2 to identify slightly longer alignments with a slightly lower overall percent identity. Stop codons are not included in the hit length, or percent identity.
• When the first Methionine (M) of a reference protein aligns to a Leucine (L), Isoleucine (I), or Valine (V) it is no-longer counted as a mismatch in the percent identity calculation reported by AMRFinderPlus. This behavior makes for more accurate statistics for proteins annotated with an incorrect early start.

At some point the PGAP GFF file output will be standardized to match other GFF files produced at NCBI; this release is to handle the small change in PGAP output. This release has no other differences from version 4.0.22, so if you're not using the --pgap or --annotation_format pgap options there is no need to update.

With the --pgap or --annotation_format pgap options AMRFinderPlus previously relied on the older non-standard format for the Name= section of the attributes field of the GFF to identify the associated protein sequence. With this update the --pgap option will parse either version of the GFF output of PGAP.

This release includes:

• A bug fix for Issue [https://github.com//issues/164] where AMRFinderPlus crashed on the combination of GZipped input files and --nucleotide_output or --protein_output.
• StxTyper version 1.0.42 which has some fixes for incomplete operons.

This version of AMRFinderPlus updates to StxTyper 1.0.40 and makes use of the --threads option to StxTyper that is new to that version. In addition we made some improvements to the tests, both at GitHub and in the included install tests.

No changes to results other than stx_operon results from StxTyper, and those changes should be minor. See the StxTyper release notes for details.

This release incorporates changes to standardize and improve the reporting format for StxTyper results in AMRFinderPlus. StxTyper is only run when the -organism Escherichia and -plus options are used and a nucleotide file is used as input. See the StxTyper release 1.0.31 for the details of these fairly minor changes in output format. There are no biologically significant differences in results with this release.

NOTE: This is a new major revision of AMRFinderPlus that has a changed database format. You will need to run amrfinder -U to get database release 2024-10-22.1 or later to use this version. This AMRFinderPlus includes StxTyper version 1.0.27 for E. coli Stx operon typing.

Software and database format changes

• See https://github.com/evolarjun/amr/wiki/New-in-AMRFinderPlus/ for more details.
• StxTyper (https://github.com/ncbi/stxtyper) which accurately types full-length stx operons now included with AMRFinderPlus.
• Database filename changes to standardize filenames and make writing scripts easier (See https://github.com/ncbi/amr/wiki/New-in-AMRFinderPlus for more information).
• Column names in output have been changed to more closely other NCBI Pathogen Detection resources such as MicroBIGG-E.
• Point mutations can now be reported with symbols different from the standard format.
• The optional hierarchy_node field will now contain two node IDs for fusion proteins and stx operons separated by '::'.
• The ability to have different curated blast cutoffs at multiple levels of the hierarchy with the same reference protein has been added.
• The same protein can be used both as a reference gene to be reported and as the reference for point mutations.

AMRFinderPlus v3.12.8 includes a new database format version. Because this version is incompatible with AMRFinderPlus databases from previous versions, amrfinder -u must be run once the software update is installed to get the latest version of the database.

This update includes the changes:

• Reference proteins now have stop codons. Input proteins that are not terminated with a ‘’ character are assumed to have a ‘’ at the end.
• Extended proteins are no longer “EXACT” hits, so the protein names will be that of the parent node for alleles.
• If a protein is hit by blast and the Blast Rule is violated, the Blast Rule of the parent family is checked, etc.
• --annotation_format prodigal input format added

This means that Previously extended hits that were 100% identical to, but longer than proteins in the database were called with the method "EXACTP" and now they are called "BLASTP". Blast results will still indicate that 100% of the database reference was covered at 100% identity, and you should still see that the "Target length" is longer than the "Reference sequence length".

1. Extended hits to allele sequences that were previously called EXACTP already had the element symbol was one node up the hierarchy (i.e. the gene symbol, not the allele symbol) the "Sequence name" was that of the ALLELE reference, and thus inconsistent with the element symbol assigned. This has been changed. Again, this does not affect previous "Element symbol" calls.

2. In combined protein + nucleotide runs of AMRFinderPlus extended protein sequences with exact matches in the database were ignored and the nucleotide match was taken resulting in an EXACTX match with no protein accession provided. Now the protein match with method BLASTP will be reported.

To reiterate, gene symbols should not change. Sequence names will only change if the query is an extended version of a protein 100% identical and completely covering an allele in the database.

Fix for rare crash and change in behavior of hierarchy_node. This release will likely have no effect for most AMRFinderPlus users.

• Fixes an unusual crash when using the --ident_min option with very low identity cutoffs (we generally don't recommend using this option).
• A change in the behavior of the --print_node option. It will now print the node associated with the element reported, rather than the most specific node hit. This changes the node reported for non-exact blast hits to reference sequences of named alleles to report the parent node. It should have no effect on all other hits and does not affect the behavior of AMRFinderPlus in any field other than hierarchy_node.