Author: Jessie Maier

# ProActive

#### Jessie Maier

# Introduction

`ProActive` automatically detects regions of gapped and elevated read coverage using a 2D pattern-matching algorithm. `ProActive` detects, characterizes and visualizes read coverage patterns in both genomes and metagenomes. Optionally, users may provide gene annotations associated with their genome or metagenome in the form of a .gff file. In this case, `ProActive` will generate an additional output table containing the gene annotations found within the detected regions of gapped and elevated read coverage. Additionally, users can search for gene annotations of interest in the output read coverage plots.

Visualizing read coverage data is important because gaps and elevations in coverage can be indicators of a variety of biological and non-biological scenarios, for example-

- Elevations and gaps in read coverage may be caused by some types of structural variants. Deletions can cause gaps while duplications can cause elevations in read coverage [1].
- Highly active and/or abundant mobile genetic elements, like transposable elements [2] and prophage [3] for example, can create elevations in read coverage at their respective integration sites.
- Genetic regions with high mutation rates and/or high variability within the population can generate gaps in read coverage [4].
- Poor quality sequencing reads and chimeric reference sequences may cause gaps and elevations in read coverage.

Since the cause for gaps and elevations in read coverage can be ambiguous, ProActive is best used as a screening method to identify genetic regions for further investigation with other tools!

References:

1. Tattini L., D’Aurizio R., & Magi A. (2015). Detection of Genomic Structural Variants from Next-Generation Sequencing Data. Frontiers in bioengineering and biotechnology, 3, 92. https://doi.org/10.3389/fbioe.2015.00092
2. Kleiner M., Bushnell B., Sanderson K.E. et al. (2020) Transductomics: sequencing-based detection and analysis of transduced DNA in pure cultures and microbial communities. Microbiome 8, 158. https://doi.org/10.1186/s40168-020-00935-5
3. Kieft K., Anantharaman K. (2022). Deciphering Active Prophages from Metagenomes. mSystems 7:e00084-22. https://doi.org/10.1128/msystems.00084-22
4. Fogarty E., Moore R. (2019). Visualizing contig coverages to better understand microbial population structure. https://merenlab.org/2019/11/25/visualizing-coverages/

# Installation

## CRAN install

    install.packages("ProActive")
    library(ProActive)

## GitHub install

    if (!require("devtools", quietly = TRUE)) {
      install.packages("devtools")
    }

    devtools::install_github("jlmaier12/ProActive")
    library(ProActive)

# Input data

## Pileups

ProActive detects read coverage patterns using a pattern-matching algorithm that operates on pileup files. A pileup file is a file format where each row summarizes the ‘pileup’ of reads at specific genomic locations. Pileup files can be used to generate a rolling mean of read coverages and associated base pair positions which reduces data size while preserving read coverage patterns. ProActive requires that input pileups files be generated using a 100 bp window/bin size.

Pileup files are generated using the .bam files produced after mapping sequencing reads to a metagenome or genome fasta file. Read mapping should be performed using a high minimum identity (0.97 or higher) and random mapping of ambiguous reads.

Some read mappers, like BBMap, allow for the generation of pileup files in the `bbmap.sh` command with use of the `bincov` output with the `covbinsize=100` parameter/argument. Otherwise, BBMap’s `pileup.sh` can convert .bam files produced by any read mapper to pileup files compatible with ProActive using the `bincov` output with `binsize=100`.

The input pileup file for metagenomes must have the following format:

Dataframe with four columns:

- V1: Contig accession
- V2: Mapped read coverage values averaged over 100 bp windows
- V3: Starting position (bp) of each 100 bp window. Restarts from 100 at the start of each new contig.
- V4: Starting position (bp) of each 100 bp window. Does NOT restart at the start of each new contig.

V1 | V2 | V3 | V4
--- | --- | --- | ---
NODE_1911 length_44214_cov_4.82142_ID_9560073 | 54.66 | 100 | 175075473
NODE_1911 length_44214_cov_4.82142_ID_9560073 | 59.13 | 200 | 175075573
NODE_1911 length_44214_cov_4.82142_ID_9560073 | 53.99 | 300 | 175075673
NODE_1911 length_44214_cov_4.82142_ID_9560073 | 54.69 | 400 | 175075773
NODE_1911 length_44214_cov_4.82142_ID_9560073 | 54.40 | 500 | 175075873
NODE_1911 length_44214_cov_4.82142_ID_9560073 | 52.11 | 600 | 175075973

Note that the format for a genome pileup will be slightly different! The third column (V3) does not restart and the fourth column (V4) starts from 0. ProActive accounts for the differences in pileup formats between genomes and metagenomes.

Users may use the ‘sampleMetagenomePileup’ and ‘sampleGenomePileup’ files that come pre-loaded with ProActive as references for proper input file format.

## gff TSV

Optionally, ProActive will accept a .gff file as additional input. The .gff file must be associated with the same metagenome or genome used to create your pileup file. The .gff file should be in TSV format and should follow the same layout described here.

The input .gff file must have the following format exactly:

V1 | V2 | V3 | V4 | V5 | V6 | V7 | V8 | V9
--- | --- | --- | --- | --- | --- | --- | --- | ---
NODE_1911 | Prodigal:002006 | CDS | 318 | 1166 | . |  | 0 | ID=NJKKNKEE_164175;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164175;product=hypothetical protein
NODE_1911 | Prodigal:002006 | CDS | 1198 | 1938 | . |  | 0 | ID=NJKKNKEE_164176;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164176;product=hypothetical protein
NODE_1911 | Prodigal:002006 | CDS | 1938 | 2582 | . |  | 0 | ID=NJKKNKEE_164177;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164177;product=hypothetical protein
NODE_1911 | Prodigal:002006 | CDS | 2722 | 3561 | . |  | 0 | ID=NJKKNKEE_164178;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164178;product=hypothetical protein
NODE_1911 | Prodigal:002006 | CDS | 3671 | 4063 | . |  | 0 | ID=NJKKNKEE_164179;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164179;product=hypothetical protein
NODE_1911 | Prodigal:002006 | CDS | 4128 | 4670 | . |  | 0 | ID=NJKKNKEE_164180;eC_number=1.11.1.1;Name=rbr1_24;db_xref=COG:COG1592;gene=rbr1_24;inference=ab initio prediction:Prodigal:002006,similar to AA sequence:UniProtKB:Q97FZ9;locus_tag=NJKKNKEE_164180;product=Rubrerythrin-1

(Hint- if you are using a gff file output by PROKKA, you may need to remove some unnecessary (for ProActive) lines of text at the top of the file. There are various ways one can remove these additional lines, however, a nice command-line solution is:)

    grep ^COMMONID metagenomeAnnots.gff > metagenomeAnnotsForProActive.gff

The ‘COMMONID’ should be a value that all of your contig or genome accessions start with. For example, the ‘COMMONID’ for the contig accessions in the sampleMetagenomegffTSV displayed above could be “NODE” since all the accessions start with “NODE”.

# ProActiveDetect()

`ProActiveDetect()` is the main function in the ProActive R package. This function filters contigs/chunks based on length and read coverage, performs pattern-matching to detect gaps and elevations in read coverage, identifies start and stop positions and sizes of pattern-matches, and, optionally, extracts gene annotations that fall within detect gaps and elevations in read coverage.

## Function components

### Chunking

Currently, `ProActiveDetect()` can only detect one gap or elevation pattern per contig. Until `ProActiveDetect()` is able to detect multiple read coverage patterns per contig, we implemented a ‘chunking’ functionality which (if `chunkContigs` = TRUE) chunks large contigs into smaller subsets (defined by `chunkSize`) so that pattern-matching can be performed on each chunk as if it were an individual contig. The chunking mechanism is what allows `ProActiveDetect()` to perform pattern-matching on entire genome sequences. When contigs/genomes are chunked, they are assigned a sequential value to link chunks back together (i.e. “NODE_1_chunk_1, NODE_1_chunk_2, NODE_1_chunk_3, …). Note that the remaining ‘chunk’ of a contig/genome may not be long enough to perform pattern-matching on. Chunks too small for pattern-matching will be put in the output FilteredOut table. If a chunk splits a gap or elevation pattern in half, `ProActiveDetect()` will attempt to detect this and report it to the user as a ‘possible pattern-match continuity’ between contig/genome chunks. Pattern-match continuity is detected when two sequential chunks have a partial elevation/gap pattern going off the right and left side of the chunks, respectively.

### Filtering

Contigs/chunks that are too short or have little to no read coverage are filtered out prior to pattern-matching. `ProActiveDetect()` filters out contigs/chunks that do not have at least 10x coverage on a total of 5,000 bp across the whole contig/chunk. The read coverage filtering was done in this way to avoid filtering out long contigs/chunks with small elevations in read coverage that might get removed if filtering was done with read coverage averages or medians. Additionally, contigs/chunks less than 30,000 bp are filtered out by default, however this can be changed with the `minContigLength` parameter which can be set to a minimum of 25,000 bp. If you would like to reduce the size of your input metagenome pileup file for `ProActiveDetect()`, consider pre-filtering your assembly for contigs greater than 25,000 bp prior to read mapping!

### Changing pileup windowSize

The input pileup files have 100 bp windows in which the mapped read coverage is averaged over. `ProActiveDetect()` increases the window size prior to pattern-matching by averaging the read coverages over a value specified with `windowSize`. In many cases, read coverage patterns don’t require the resolution that 100 bp windows provide, however, starting with a 100 bp windowSize means the higher resolution is available if needed. While users can use the 100 bp `windowSize` for `ProActiveDetect()`, the processing time will be increased significantly and noisy data may interfere with pattern-matching. We find that the default 1,000 bp `windowSize` provides a nice balance between processing time and read coverage pattern resolution. If you’d like more resolution than the 1,000 bp `windowSize` provides, consider dropping the `windowSize` to 500. If you’d like fine scale read coverage resolution, consider viewing the contigs/genome with a software like Integrative Genomics Viewer IGV.

### Pattern-matching

`ProActiveDetect()` detects read coverage patterns using a 2D pattern-matching algorithm. Several predefined patterns, described below, are built using the specific length and read coverage values of the contig/chunk being assessed. Patterns are translated across each contig/chunk in 1,000 bp sliding windows and at each translation, a pattern-match score is calculated by taking the mean absolute difference of the read coverage and the pattern values. The smaller the match-score, the better the pattern-match. After a pattern is fully translated across a contig/chunk, certain aspects of the pattern are changed (i.e. height, base, width) and translation is repeated. This process of translation and pattern re-scaling is repeated until a large number of pattern variations are tested. After pattern-matching is complete, the pattern associated with the best match-score is used for contig/chunk classification. Contigs/chunks are classified as ‘Elevation’, ‘Gap’, or ‘NoPattern’ during pattern-matching.

#### Elevation pattern:

The ‘elevation’ class is defined by a ‘block’ pattern. During pattern-matching, the height (max.), base (min.) and width are altered and all pattern variations are translated across the contig/chunk. The block width never gets smaller than 10,000 bp by default, however this can be changed with the `minSize` parameter.

#### Gap pattern:

The ‘gap’ class is essentially the reverse of the values used to build the block pattern in the ‘elevation’ class. The same pattern-matching steps (alteration of pattern max., min. and width values and pattern translation) used for the elevation pattern are used for the gap pattern.

#### Elevation/Gap pattern:

Elevations and gaps that trail off one side of a contig/chunk are hard to classify as the read coverage can be interpreted as a gap or elevation depending on how you’re looking at it. We classify contigs/chunk as ‘Gap’ if the elevated region is greater than 50% of the length of the contig/chunk. Conversely, if the elevated region is less than 50% of the contig/chunk length, the classification is ‘Elevation’.

#### noPattern:

Since the best pattern-match for each contig/chunk is determined by comparing match-scores amongst all pattern-variations from all pattern classes, we needed a ‘negative control’ pattern to compare against. The ‘NoPattern’ ‘pattern’ serves as a negative control by matching to contigs/chunks with no read coverage patterns. We made two NoPattern patterns which consist of a horizontal line the same length as the contig/chunk being assessed at the contig/chunk’s average and median read coverage value. This pattern is not re-scaled or translated in any way.

### Calculating elevation ratios

Every gap and elevation classification receives an ‘elevation ratio’ value which is simply the pattern-match’s maximum value divided by the minimum value. For Elevation classifications, you can think of the elevation ratio as how many times greater the read coverage of the elevated region is compare to the non-elevated region’. Conversely, for Gap classifications, the elevation ratio is how many times less the read coverage of the gap region is compared to the non-gapped region.

### Extracting gene annotations in elevated/gapped regions

If a .gff file is provided, then `ProActiveDetect()` will extract the gene annotations found within the gapped and elevated pattern-match regions and provide them to the user in an output table (GeneAnnotTable). An additional column will be added with the classification information (Gap or Elevation) associated with the gene annotations. If the input .gff file contains a gene ‘product’ field in the attributes column (9th column in the dataframe), then `ProActiveDetect()` will extract the product information into a separate column for easy visualization and filtering of annotations of interest.

## Usage

Default arguments in metagenome mode:

    ProActiveOutputMetagenome <- ProActiveDetect(
      pileup = sampleMetagenomePileup,
      mode = "metagenome",
      gffTSV = sampleMetagenomegffTSV
    )
    #> Preparing input file for pattern-matching...
    #> Starting pattern-matching...
    #> A quarter of the way done with pattern-matching
    #> Half of the way done with pattern-matching
    #> Almost done with pattern-matching!
    #> Summarizing pattern-matching results
    #> Finding gene predictions in elevated or gapped regions of read coverage...
    #> Finalizing output
    #> Execution time: 3.32secs
    #> 0 contigs were filtered out based on low read coverage
    #> 0 contigs were filtered out based on length (< minContigLength)
    #>
    #> Elevation       Gap NoPattern
    #>         3         3         1

Default arguments in genome mode:

    ProActiveOutputGenome <- ProActiveDetect(
      pileup = sampleGenomePileup,
      mode = "genome",
      gffTSV = sampleGenomegffTSV
    )
    #> Preparing input file for pattern-matching...
    #> Starting pattern-matching...
    #> A quarter of the way done with pattern-matching
    #> Half of the way done with pattern-matching
    #> Almost done with pattern-matching!
    #> Summarizing pattern-matching results
    #> Finding gene predictions in elevated or gapped regions of read coverage...
    #> Finalizing output
    #> Execution time: 43.67secs
    #> 0 contigs were filtered out based on low read coverage
    #> 0 contigs were filtered out based on length (< minContigLength)
    #>
    #> Elevation       Gap NoPattern
    #>        25         3        21

Note that `ProActiveDetect()` can be run without the gffTSV file!

## Arguments/parameters

    ProActiveDetect(
      pileup,
      mode,
      gffTSV,
      windowSize = 1000,
      minSize = 10000,
      maxSize = Inf,
      minContigLength = 30000,
      chunkSize = 50000,
      chunkContigs = FALSE,
      IncludeNoPatterns = FALSE,
      verbose = TRUE,
      saveFilesTo
    )

- `pileup`: A .txt file containing mapped sequencing read coverages averaged over 100 bp windows/bins.
- `mode`: Either “genome” or “metagenome”.
- `gffTSV`: Optional, a .gff file (TSV) containing gene annotations associated with the .fasta file used to generate the pileup.
- `windowSize`: The number of basepairs to average read coverage values over. Options are 100, 200, 500, 1000 ONLY. Default is 1000.
- `minSize`: The minimum size (in bp) of elevation or gap patterns. Default is 10000.
- `maxSize`: The maximum size (in bp) of elevation or gap patterns. Default is NA (i.e. no maximum).
- `minContigLength`: The minimum contig/chunk size (in bp) to perform pattern-matching on. Default is 25000.
- `chunkSize`: If `mode = "genome"` OR if `mode = "metagenome"` and `chunkContigs = TRUE`, chunk the genome or contigs, respectively, into smaller subsets for pattern-matching. `chunkSize` determines the size (in bp) of each ‘chunk’. Default is 100000.
- `chunkContigs`: TRUE or FALSE, If TRUE and `mode = "metagenome"`, contigs longer than the `chunkSize` will be ‘chunked’ into smaller subsets and pattern-matching will be performed on each subset. Default is FALSE.
- `IncludeNoPatterns`: TRUE or FALSE, If TRUE the noPattern pattern-matches will be included in the PatternMatches output list. If you would like to visualize the read coverage of noPattern classifications in `plotProActiveResults()`, this should be set to TRUE.
- `verbose`: TRUE or FALSE. Print progress messages to console. Default is TRUE.
- `saveFilesTo`: Optional, Provide a path to the directory you wish to save output to. A folder will be made within the provided directory to store results.

## Output

The output of `ProActiveDetect()` is a list containing six objects:

1. SummaryTable: A table containing all pattern-matching classifications
2. CleanSummaryTable: A table containing only Gap and Elevation pattern-match classifications (i.e. noPattern classifications removed)
3. PatternMatches: A list object containing information needed to visualize the pattern-matches in `plotProActiveResults()`
4. FilteredOut: A table containing contigs/chunks that were filtered out for being too small or having too low read coverage
5. Arguments: A list object containing arguments used for pattern-matching (windowSize, mode, chunkSize, chunkContigs)
6. GeneAnnotTable: A table containing gene annotations associated with elevated or gapped regions in pattern-matches

Save the desired list item to a new variable using its associated name.

Metagenome results summary table:

    MetagenomeCleanSummaryTable <- ProActiveOutputMetagenome$CleanSummaryTable

refName | classification | elevRatio | startPos | endPos | matchSize
--- | --- | --- | --- | --- | ---
NODE_1911 | Elevation | 3.349296 | 1000 | 17000 | 16000
NODE_1583 | Elevation | 2.450013 | 42000 | 51000 | 9000
NODE_1884 | Gap | 3.319514 | 36000 | 44000 | 8000
NODE_1255 | Gap | 5.318072 | 1000 | 20000 | 19000
NODE_368 | Gap | 2.690172 | 26000 | 56000 | 30000
NODE_617 | Elevation | 1.784556 | 34000 | 82000 | 48000

Subset of genome results summary table:

    GenomeCleanSummaryTable <- head(ProActiveOutputGenome$CleanSummaryTable)

| refName | classification | elevRatio | startPos | endPos | matchSize
--- | --- | --- | --- | --- | --- | ---
3 | NC_003197.2_chunk_3 | Gap | 1.609512 | 283000 | 300000 | 17000
4 | NC_003197.2_chunk_4 | Elevation | 1.351684 | 339000 | 351000 | 12000
7 | NC_003197.2_chunk_7 | Elevation | 1.343521 | 692000 | 700000 | 8000
8 | NC_003197.2_chunk_8 | Elevation | 1.723227 | 784000 | 794000 | 10000
10 | NC_003197.2_chunk_10 | Elevation | 1.887571 | 964000 | 1000000 | 36000
12 | NC_003197.2_chunk_12 | Elevation | 1.293144 | 1134000 | 1144000 | 10000

Subset of GeneAnnotTable for metagenome results:

    MetagenomeResultsGenePredictTable <- head(ProActiveOutputMetagenome$GeneAnnotTable)

seqid | source | type | start | end | score | strand | phase | attributes | geneproduct | Classification
--- | --- | --- | --- | --- | --- | --- | --- | --- | --- | ---
NODE_1911 | Prodigal:002006 | CDS | 318 | 1166 | . |  | 0 | ID=NJKKNKEE_164175;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164175;product=hypothetical protein | hypothetical protein | Elevation
NODE_1911 | Prodigal:002006 | CDS | 1198 | 1938 | . |  | 0 | ID=NJKKNKEE_164176;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164176;product=hypothetical protein | hypothetical protein | Elevation
NODE_1911 | Prodigal:002006 | CDS | 1938 | 2582 | . |  | 0 | ID=NJKKNKEE_164177;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164177;product=hypothetical protein | hypothetical protein | Elevation
NODE_1911 | Prodigal:002006 | CDS | 2722 | 3561 | . |  | 0 | ID=NJKKNKEE_164178;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164178;product=hypothetical protein | hypothetical protein | Elevation
NODE_1911 | Prodigal:002006 | CDS | 3671 | 4063 | . |  | 0 | ID=NJKKNKEE_164179;inference=ab initio prediction:Prodigal:002006;locus_tag=NJKKNKEE_164179;product=hypothetical protein | hypothetical protein | Elevation
NODE_1911 | Prodigal:002006 | CDS | 4128 | 4670 | . |  | 0 | ID=NJKKNKEE_164180;eC_number=1.11.1.1;Name=rbr1_24;db_xref=COG:COG1592;gene=rbr1_24;inference=ab initio prediction:Prodigal:002006,similar to AA sequence:UniProtKB:Q97FZ9;locus_tag=NJKKNKEE_164180;product=Rubrerythrin-1 | Rubrerythrin-1 | Elevation

Subset of GeneAnnotTable for genome results:

    GenomeResultsGenePredictTable <- head(ProActiveOutputGenome$GeneAnnotTable)

seqid | source | type | start | end | score | strand | phase | attributes | geneproduct | Classification
--- | --- | --- | --- | --- | --- | --- | --- | --- | --- | ---
NC_003197.2_chunk_3 | Prodigal:002006 | CDS | 282468 | 284186 | . |  | 0 | ID=LFLNNMPD_00240;eC_number=6.1.1.15;Name=proS;db_xref=COG:COG0442;gene=proS;inference=ab initio prediction:Prodigal:002006,similar to AA sequence:UniProtKB:P16659;locus_tag=LFLNNMPD_00240;product=Proline–tRNA ligase | Proline–tRNA ligase | Gap
NC_003197.2_chunk_3 | Prodigal:002006 | CDS | 284297 | 285004 | . |  | 0 | ID=LFLNNMPD_00241;eC_number=2.1.1.-;Name=trmO;db_xref=COG:COG1720;gene=trmO;inference=ab initio prediction:Prodigal:002006,similar to AA sequence:UniProtKB:P28634;locus_tag=LFLNNMPD_00241;product=tRNA (adenine(37)-N6)-methyltransferase | tRNA (adenine(37)-N6)-methyltransferase | Gap
NC_003197.2_chunk_3 | Prodigal:002006 | CDS | 285001 | 285405 | . |  | 0 | ID=LFLNNMPD_00242;Name=rcsF;gene=rcsF;inference=ab initio prediction:Prodigal:002006,similar to AA sequence:UniProtKB:P69411;locus_tag=LFLNNMPD_00242;product=Outer membrane lipoprotein RcsF | Outer membrane lipoprotein RcsF | Gap
NC_003197.2_chunk_3 | Prodigal:002006 | CDS | 285524 | 286339 | . |  | 0 | ID=LFLNNMPD_00243;Name=metQ_1;db_xref=COG:COG1464;gene=metQ_1;inference=ab initio prediction:Prodigal:002006,similar to AA sequence:UniProtKB:P28635;locus_tag=LFLNNMPD_00243;product=D-methionine-binding lipoprotein MetQ | D-methionine-binding lipoprotein MetQ | Gap
NC_003197.2_chunk_3 | Prodigal:002006 | CDS | 286378 | 287031 | . |  | 0 | ID=LFLNNMPD_00244;Name=metI;db_xref=COG:COG2011;gene=metI;inference=ab initio prediction:Prodigal:002006,similar to AA sequence:UniProtKB:P31547;locus_tag=LFLNNMPD_00244;product=D-methionine transport system permease protein MetI | D-methionine transport system permease protein MetI | Gap
NC_003197.2_chunk_3 | Prodigal:002006 | CDS | 287024 | 288055 | . |  | 0 | ID=LFLNNMPD_00245;eC_number=3.6.3.-;Name=metN_1;db_xref=COG:COG1135;gene=metN_1;inference=ab initio prediction:Prodigal:002006,similar to AA sequence:UniProtKB:P30750;locus_tag=LFLNNMPD_00245;product=Methionine import ATP-binding protein MetN | Methionine import ATP-binding protein MetN | Gap

# plotProActiveResults()

`plotProActiveResults()` allows users to visualize both the read coverage and the pattern-match associated with each Gap or Elevation classification.

## Function components

### Re-building pattern-matches

The `ProActiveDetect()` output contains information needed to re-build each pattern-match used for classification. To re-build a complete pattern-match for visualization, `plotProActiveResults()` uses the pattern-match’s minimum and maximum values and the start and stop positions.

### Plotting read coverage and associated pattern-matches

By default, the read coverage is plotted for each contig/chunk classified as having a Gap or Elevation in read coverage. If you wish to see the read coverage for noPattern classifications, be sure to set `IncludeNoPatterns = TRUE` when running `ProActiveDetect()`. The pattern-match associated with each classification is overlaid on the coverage plot.

## Usage

Default arguments:

    MetagenomeResultsPlots <- plotProActiveResults(
      pileup = sampleMetagenomePileup,
      ProActiveResults = ProActiveOutputMetagenome
    )

    GenomeResultsPlots <- plotProActiveResults(
      pileup = sampleGenomePileup,
      ProActiveResults = ProActiveOutputGenome
    )

Note- There is no need to set ‘genome’ or ‘metagenome’ mode. `plotProActiveResults()` will get this information from the `ProActiveDetect()` output.

## Arguments/parameters

    plotProActiveResults(pileup,
      ProActiveResults,
      elevFilter,
      saveFilesTo
    )

- `pileup`: A .txt file containing mapped sequencing read coverages averaged over 100 bp windows/bins.
- `ProActiveResults`: The output from `ProActiveDetect()`.
- `elevFilter`: Optional, only plot results with pattern-matches that achieved an elevation ratio (max/min) greater than the specified value. Default is no filter.
- `saveFilesTo`: Optional, Provide a path to the directory you wish to save output to. A folder will be made within the provided directory to store results.

## Output

The output of `plotProActiveResults()` is a list of ggplot objects.

### View select metagenome plots

    MetagenomeResultsPlots$NODE_1884

    MetagenomeResultsPlots$NODE_368

    MetagenomeResultsPlots$NODE_617

### View select genome plots

Notice the ‘chunk’ information in the plot titles

    GenomeResultsPlots$NC_003197.2_chunk_36

    GenomeResultsPlots$NC_003197.2_chunk_8

# geneAnnotationSearch()

`geneAnnotationSearch()` helps users explore gene annotations of interest in and around detected gaps and elevations in read coverage.

## Function components

### Search for gene annotations

`geneAnnotationSearch()` utilizes a .gff file and the pattern-matching results from `ProActiveDetect()` to locate gene annotations that match provided `keyWords`. The .gff file should be in the same format described previously in the Input Files section of this vignette. First, the information associated with the gene or gene product (depending on what the user selects for the `geneOrProduct` parameter) is extracted from the attributes column of the .gff file. Then, the .gff file is subset to include only the annotations associated with the contig/chunk being assessed. From here, the search can vary quite a bit depending on the parameters the user selects for the `inGapOrElev` and `bpRange` parameters. If `inGapOrElev = FALSE` (the default), then gene annotations located anywhere on the contig/chunk that match one or more of the provided `keyWords` will be visualized. If `inGapOrElev = TRUE`, then only gene annotations within the gap/elevation region of the pattern-match will be searched for matches to the provided `keyWords`. The `bpRange` parameter can be used if `inGapOrElev = TRUE` and allows the search range to be extended a specified number of base pairs to the left and right of the gap/elevation pattern-match borders. Gene annotation are included in the search if the end of the open reading frame (defined by the ‘end’ values in the .gff file) falls within the search region.

### Plot gene annotation locations

The read coverage and locations of gene annotations that match the provided `keyWords` are visualized for each contig/chunk with matches. The read coverage is plotted using a 100 bp windowSize to allow for greater resolution of read coverage patterns and gene annotation locations. The borders of the elevated/gapped regions of read coverage detected by `ProActiveDetect()` are marked on the plot with orange vertical lines. If `inElevOrGap = TRUE` and `bpRange` is set to a non-zero value, then the extended search range outside the gap/elevation borders are marked on the plot with orange dashed vertical lines. The matching gene annotation locations are marked on the plot with black vertical lines at the start position of the associated open reading frames.

Note- The pattern-matching used to identify the borders of elevated and gapped regions of read coverage was likely performed using a `windowSize` larger than 100 bp in `ProActiveDetect()`. This means that the locations of the borders may not perfectly translate to the borders of gaps and elevations at 100 bp resolution.

## Usage

Default arguments:

With defaults, all contigs/chunks classified as having a gap or elevation in read coverage are searched for gene annotations that match any of the provided keywords. The entire contig/chunk is searched, not just the gapped or elevated region.

    MetagenomeGeneMatches <- geneAnnotationSearch(
      ProActiveResults = ProActiveOutputMetagenome,
      pileup = sampleMetagenomePileup,
      gffTSV = sampleMetagenomegffTSV,
      geneOrProduct = "product",
      keyWords = c("transport", "chemotaxis")
      )
    #> Cleaning gff file...
    #> Cleaning pileup file...
    #> Searching for matching annotations...
    #> 3 contigs/chunks have gene annotations that match one or more of the provided keyWords

Non-default arguments

With the following parameters/arguments, all classified contigs/chunks are searched for gene annotations that match the provided keywords (same as default), BUT with the use of `inGapOrElev = TRUE`, only the gapped or elevated region is searched for matching annotations. Additionally, the use of `bpRange = 5000` means that the search region is extended 5,000 bp from the left and right of the gapped or elevated region.

    GenomeGeneMatches <- geneAnnotationSearch(
      ProActiveResults = ProActiveOutputGenome,
      pileup =  sampleGenomePileup,
      gffTSV = sampleGenomegffTSV,
      geneOrProduct = "product",
      keyWords = c("ribosomal"),
      inGapOrElev = TRUE,
      bpRange = 5000
      )
    #> Cleaning gff file...
    #> Cleaning pileup file...
    #> Searching for matching annotations...
    #> 8 contigs/chunks have gene annotations that match one or more of the provided keyWords

## Arguments/parameters

    geneAnnotationSearch(
      ProActiveResults,
      pileup,
      gffTSV,
      geneOrProduct,
      keyWords,
      inGapOrElev = FALSE,
      bpRange = 0,
      elevFilter,
      saveFilesTo,
      verbose = TRUE
      )

- `ProActiveResults`: The output from `ProActiveDetect()`.
- `pileup`: A .txt file containing mapped sequencing read coverages averaged over 100 bp windows/bins.
- `gffTSV`: A .gff file (TSV) containing gene annotations associated with the .fasta file used to generate the pileup.
- `geneOrProduct`: “gene” or “product”. Search for keyWords associated with genes or gene products.
- `keyWords`: The keyWord(s) to search for. Case independent. Searches will return the string #’ that contains the matching keyWord. KeyWord(s) must be in quotes, comma-separated, and surrounded by #’ c() i.e( c(“antibiotic”, “resistance”, “drug”) )
- `inGapOrElev`: TRUE or FALSE. If TRUE, only search for gene-annotations in #’ the gap/elevation region of the pattern-match. Default is FALSE (i.e search the #’ entire contig/chunk for the gene annotation key-words)
- `bpRange`: If `inGapOrElev = TRUE`, the user may specify the region (in base pairs) that should #’ be searched to the left and right of the gap/elevation region. Default is 0.
- `elevFilter`: Optional, only plot results with pattern-matches that achieved an elevation ratio (max/min) greater than the specified value. Default is no filter.
- `saveFilesTo`: Optional, Provide a path to the directory you wish to save output to. A folder will be made within the provided directory to store results.
- `verbose`: TRUE or FALSE. Print progress messages to console. Default is TRUE.

## Output

The output of `geneAnnotationSearch()` is a list of ggplot objects.

Default search parameters:

    MetagenomeGeneMatches$NODE_617

Non-default search parameters (use of `inGapOrElev` = TRUE and `bpRange` = 5000)

    GenomeGeneMatches$NC_003197.2_chunk_3

    GenomeGeneMatches$NC_003197.2_chunk_36

# Session Information

    sessionInfo()
    #> R version 4.2.1 (2022-06-23 ucrt)
    #> Platform: x86_64-w64-mingw32/x64 (64-bit)
    #> Running under: Windows 10 x64 (build 19045)
    #>
    #> Matrix products: default
    #>
    #> locale:
    #> [1] LC_COLLATE=C
    #> [2] LC_CTYPE=English_United States.utf8
    #> [3] LC_MONETARY=English_United States.utf8
    #> [4] LC_NUMERIC=C
    #> [5] LC_TIME=English_United States.utf8
    #>
    #> attached base packages:
    #> [1] stats     graphics  grDevices utils     datasets  methods   base
    #>
    #> other attached packages:
    #> [1] dplyr_1.1.4      stringr_1.5.1    ggplot2_3.5.1    kableExtra_1.4.0
    #> [5] ProActive_0.1.0
    #>
    #> loaded via a namespace (and not attached):
    #>  [1] bslib_0.8.0       compiler_4.2.1    pillar_1.9.0      jquerylib_0.1.4
    #>  [5] tools_4.2.1       digest_0.6.37     viridisLite_0.4.2 jsonlite_1.8.9
    #>  [9] evaluate_1.0.1    lifecycle_1.0.4   tibble_3.2.1      gtable_0.3.6
    #> [13] pkgconfig_2.0.3   rlang_1.1.4       cli_3.6.2         rstudioapi_0.17.1
    #> [17] yaml_2.3.10       xfun_0.49         fastmap_1.2.0     withr_3.0.2
    #> [21] xml2_1.3.6        knitr_1.49        systemfonts_1.1.0 generics_0.1.3
    #> [25] vctrs_0.6.5       sass_0.4.9        grid_4.2.1        tidyselect_1.2.1
    #> [29] svglite_2.1.3     glue_1.8.0        R6_2.5.1          fansi_1.0.6
    #> [33] rmarkdown_2.29    farver_2.1.2      magrittr_2.0.3    scales_1.3.0
    #> [37] htmltools_0.5.8.1 colorspace_2.1-1  labeling_0.4.3    utf8_1.2.4
    #> [41] stringi_1.8.4     munsell_0.5.1     cachem_1.1.0