added 2 QC thresholds to ANI task to reduce false positives

tasks/quality_control/task_mummer_ani.wdl

@@ -6,12 +6,17 @@ task animummer {
     String samplename
     File? ref_genome
     Float mash_filter = 0.9
-    String docker="us-docker.pkg.dev/general-theiagen/staphb/mummer:4.0.0-rgdv2"
+    # these 2 thresholds were set as they are used by CDC enterics lab/PulseNet for ANI thresholds
+    Float ani_threshold = 80.0
+    Float percent_bases_aligned_threshold = 70.0
+    String docker= "us-docker.pkg.dev/general-theiagen/staphb/mummer:4.0.0-rgdv2"
+    Int cpus = 4
+    Int memory = 8
     Int disk_size = 100
   }
   command <<<
     # capture and version
-    mummer --version | tee MUMMER_VERSION
+    mummer --version | tee MUMMER_VERSION.txt
 
     # set the reference genome
     # if not defined by user, then use all 43 genomes in RGDv2
@@ -42,18 +47,22 @@ task animummer {
       echo "~{samplename} did not surpass the minimum mash genetic distance filter, thus ANI was not performed"
       echo "The output TSV only contains the header line"
       # set output variables as 0s or descriptive strings
-      echo "0.0" > ANI_HIGHEST_PERCENT_BASES_ALIGNED
-      echo "0.0" > ANI_HIGHEST_PERCENT
-      echo "ANI skipped due to high genetic divergence from reference genomes" > ANI_TOP_SPECIES_MATCH
+      echo "0.0" > ANI_HIGHEST_PERCENT_BASES_ALIGNED.txt
+      echo "0.0" > ANI_HIGHEST_PERCENT.txt
+      echo "ANI skipped due to high genetic divergence from reference genomes" > ANI_TOP_SPECIES_MATCH.txt
     # if output TSV has greater than 1 lines, then parse for appropriate outputs
     else
       ## parse out highest percentBases aligned
-      cut -f 5 ~{samplename}.ani-mummer.out.tsv | sort -nr | head -n 1 | tee ANI_HIGHEST_PERCENT_BASES_ALIGNED
-      echo "highest percent bases aligned is: $(cat ANI_HIGHEST_PERCENT_BASES_ALIGNED)"
+      cut -f 5 ~{samplename}.ani-mummer.out.tsv | sort -nr | head -n 1 | tee ANI_HIGHEST_PERCENT_BASES_ALIGNED.txt
+      echo "highest percent bases aligned is: $(cat ANI_HIGHEST_PERCENT_BASES_ALIGNED.txt)"
+      ANI_HIGHEST_PERCENT_BASES_ALIGNED=$(cat ANI_HIGHEST_PERCENT_BASES_ALIGNED.txt)
 
       ## parse out ANI value using highest percentBases aligned value
-      grep "$(cat ANI_HIGHEST_PERCENT_BASES_ALIGNED)" ~{samplename}.ani-mummer.out.tsv | cut -f 3 | tee ANI_HIGHEST_PERCENT
-      echo "ANI value is: $(cat ANI_HIGHEST_PERCENT)"
+      grep "$(cat ANI_HIGHEST_PERCENT_BASES_ALIGNED.txt)" ~{samplename}.ani-mummer.out.tsv | cut -f 3 | tee ANI_HIGHEST_PERCENT.txt
+      echo "Highest ANI value is: $(cat ANI_HIGHEST_PERCENT.txt)"
+      # set ANI_HIGHEST_PERCENT as a bash variable (float)
+      ANI_HIGHEST_PERCENT=$(cat ANI_HIGHEST_PERCENT.txt)
+
 
       # have to separate out results for ani_top_species match because user-defined reference genome FASTAs will not be named as they are in RGDv2
       if [[ -z "~{ref_genome}" ]]; then
@@ -63,27 +72,46 @@ task animummer {
         # cut on periods to pull out genus_species (in future this will inlcude lineages for Listeria and other sub-species designations)
         # have to create assembly_file_basename bash variable since output TSV does not include full path to assembly file, only filename
         assembly_file_basename=$(basename ~{assembly})
-        grep "$(cat ANI_HIGHEST_PERCENT)" ~{samplename}.ani-mummer.out.tsv | cut -f 1,2 | sed "s|${assembly_file_basename}||g" | xargs | cut -d '.' -f 3 | tee ANI_TOP_SPECIES_MATCH
-        echo "ANI top species match is: $(cat ANI_TOP_SPECIES_MATCH)"
+        grep "${ANI_HIGHEST_PERCENT}" ~{samplename}.ani-mummer.out.tsv | cut -f 1,2 | sed "s|${assembly_file_basename}||g" | xargs | cut -d '.' -f 3 | tee ANI_TOP_SPECIES_MATCH.txt
+        echo "ANI top species match is: $(cat ANI_TOP_SPECIES_MATCH.txt)"
+
+        # if ANI threshold or percent_bases_aligned_threshold is defined by user (they both are by default), compare to highest ANI value and corresponding percent_bases_aligned value and only output ANI_top_species_match if both thresholds are surpassed
+        if [[ -n "~{ani_threshold}" || -n "~{percent_bases_aligned_threshold}" ]]; then
+          echo "Comparing user-defined ANI threshold to highest ANI value..."
+          # compare ANI_HIGHEST_PERCENT to ani_threshold using awk
+          if ! awk "BEGIN{ exit ($ANI_HIGHEST_PERCENT < ~{ani_threshold} )}"; then
+            echo "The highest ANI value $ANI_HIGHEST_PERCENT is less than the user-defined ANI threshold of ~{ani_threshold}"
+            echo "ANI top species match did not surpass the user-defined ANI threshold of ~{ani_threshold}" > ANI_TOP_SPECIES_MATCH.txt
+          # else if: compare percent_bases_aligned_threshold to ANI_HIGHEST_PERCENT_BASES_ALIGNED using awk
+          elif ! awk "BEGIN{ exit (${ANI_HIGHEST_PERCENT_BASES_ALIGNED} < ~{percent_bases_aligned_threshold} )}"; then
+            echo "The highest ANI percent bases aligned value ${ANI_HIGHEST_PERCENT_BASES_ALIGNED} is less than the user-defined threshold of ~{percent_bases_aligned_threshold}"
+            # overwrite ANI_TOP_SPECIES_MATCH.txt when percent_bases_aligned threshold is not surpassed
+            echo "ANI top species match did not surpass the user-defined percent bases aligned threshold of ~{percent_bases_aligned_threshold}" > ANI_TOP_SPECIES_MATCH.txt 
+          else
+            echo "The highest ANI value ${ANI_HIGHEST_PERCENT} is greater than the user-defined threshold ~{ani_threshold}"
+            echo "The highest percent bases aligned value ${ANI_HIGHEST_PERCENT_BASES_ALIGNED} is greater than the user-defined threshold ~{percent_bases_aligned_threshold}"
+          fi
+        fi
       else 
         # User specified a reference genome, use fasta filename as output string
-        basename "${REF_GENOME}" > ANI_TOP_SPECIES_MATCH
+        basename "${REF_GENOME}" > ANI_TOP_SPECIES_MATCH.txt
         echo "Reference genome used for ANI is: ${REF_GENOME}"
       fi
     fi
 
   >>>
   output {
-    Float ani_highest_percent = read_float("ANI_HIGHEST_PERCENT")
-    Float ani_highest_percent_bases_aligned = read_float("ANI_HIGHEST_PERCENT_BASES_ALIGNED")
+    Float ani_highest_percent = read_float("ANI_HIGHEST_PERCENT.txt")
+    Float ani_highest_percent_bases_aligned = read_float("ANI_HIGHEST_PERCENT_BASES_ALIGNED.txt")
     File ani_output_tsv = "~{samplename}.ani-mummer.out.tsv"
-    String ani_top_species_match = read_string("ANI_TOP_SPECIES_MATCH")
-    String ani_mummer_version = read_string("MUMMER_VERSION")
+    String ani_top_species_match = read_string("ANI_TOP_SPECIES_MATCH.txt")
+    String ani_mummer_version = read_string("MUMMER_VERSION.txt")
+    String ani_docker = "~{docker}"
   }
   runtime {
     docker:  "~{docker}"
-    memory:  "8 GB"
-    cpu:   4
+    memory:  "~{memory} GB"
+    cpu:  cpus
     disks: "local-disk " + disk_size + " SSD"
     disk: disk_size + " GB"
     maxRetries: 3

tasks/utilities/task_broad_terra_tools.wdl

@@ -92,6 +92,7 @@ task export_taxon_tables {
     File? ani_output_tsv
     String? ani_top_species_match 
     String? ani_mummer_version
+    String? ani_docker
     String? kmerfinder_docker
     File? kmerfinder_results_tsv
     String? kmerfinder_top_hit
@@ -595,6 +596,7 @@ task export_taxon_tables {
       "ani_output_tsv": "~{ani_output_tsv}",
       "ani_top_species_match": "~{ani_top_species_match}",
       "ani_mummer_version": "~{ani_mummer_version}",
+      "ani_docker": "~{ani_docker}",
       "kmerfinder_docker": "~{kmerfinder_docker}",
       "kmerfinder_results_tsv": "~{kmerfinder_results_tsv}",
       "kmerfinder_top_hit": "~{kmerfinder_top_hit}",

tests/workflows/theiaprok/test_wf_theiaprok_illumina_pe.yml

@@ -574,7 +574,7 @@
     - path: miniwdl_run/wdl/tasks/quality_control/task_fastq_scan.wdl
       contains: ["version", "fastq_scan", "output"]
     - path: miniwdl_run/wdl/tasks/quality_control/task_mummer_ani.wdl
-      md5sum: 21e8b1cbe4c8be26d1ac8b8013970166
+      md5sum: d50a40d533d51834d0000971dc2c2014
     - path: miniwdl_run/wdl/tasks/quality_control/task_ncbi_scrub.wdl
       contains: ["version", "scrub", "output"]
     - path: miniwdl_run/wdl/tasks/quality_control/task_qc_check_phb.wdl
@@ -624,17 +624,17 @@
     - path: miniwdl_run/wdl/tasks/species_typing/task_ts_mlst.wdl
       md5sum: d49ae0b02e798af0636eb2721bb434b4
     - path: miniwdl_run/wdl/tasks/task_versioning.wdl
-      md5sum: 9c02b95d23a602d9842fdeac883037b1
+      md5sum: 3469cf6787f279ffa81fa50f6257fcd7
     - path: miniwdl_run/wdl/tasks/taxon_id/task_gambit.wdl
       md5sum: 08987d952c67c6ff6debf6898af15f9a
     - path: miniwdl_run/wdl/tasks/taxon_id/task_kraken2.wdl
       md5sum: a1f287e6e6feaf2d7d3c74a70e3b5a28
     - path: miniwdl_run/wdl/tasks/taxon_id/task_midas.wdl
       md5sum: 024971d1439dff7d59c0a26a824bd2c6
     - path: miniwdl_run/wdl/tasks/utilities/task_broad_terra_tools.wdl
-      md5sum: 43ef050bde1fb8755f38e697a1794918
+      md5sum: 26ac9a20c8043a28d373bfe0ca361cc6
     - path: miniwdl_run/wdl/workflows/theiaprok/wf_theiaprok_illumina_pe.wdl
-      md5sum: 0c87a7279c4870a821c3dc1db9a6a94b
+      md5sum: ac4971ad992c3e8abee5d3817928a8f2
     - path: miniwdl_run/wdl/workflows/utilities/wf_merlin_magic.wdl
       md5sum: 00bd2489b2a7aa5b88340a940961a857
     - path: miniwdl_run/wdl/workflows/utilities/wf_read_QC_trim_pe.wdl

tests/workflows/theiaprok/test_wf_theiaprok_illumina_se.yml

@@ -544,7 +544,7 @@
     - path: miniwdl_run/wdl/tasks/quality_control/task_fastq_scan.wdl
       contains: ["version", "fastq_scan", "output"]
     - path: miniwdl_run/wdl/tasks/quality_control/task_mummer_ani.wdl
-      md5sum: 21e8b1cbe4c8be26d1ac8b8013970166
+      md5sum: d50a40d533d51834d0000971dc2c2014
     - path: miniwdl_run/wdl/tasks/quality_control/task_qc_check_phb.wdl
       contains: ["version", "qc_check_table", "output"]
     - path: miniwdl_run/wdl/tasks/quality_control/task_quast.wdl
@@ -592,17 +592,17 @@
     - path: miniwdl_run/wdl/tasks/species_typing/task_ts_mlst.wdl
       md5sum: d49ae0b02e798af0636eb2721bb434b4
     - path: miniwdl_run/wdl/tasks/task_versioning.wdl
-      md5sum: 9c02b95d23a602d9842fdeac883037b1
+      md5sum: 3469cf6787f279ffa81fa50f6257fcd7
     - path: miniwdl_run/wdl/tasks/taxon_id/task_gambit.wdl
       md5sum: 08987d952c67c6ff6debf6898af15f9a
     - path: miniwdl_run/wdl/tasks/taxon_id/task_kraken2.wdl
       md5sum: a1f287e6e6feaf2d7d3c74a70e3b5a28
     - path: miniwdl_run/wdl/tasks/taxon_id/task_midas.wdl
       md5sum: 024971d1439dff7d59c0a26a824bd2c6
     - path: miniwdl_run/wdl/tasks/utilities/task_broad_terra_tools.wdl
-      md5sum: 43ef050bde1fb8755f38e697a1794918
+      md5sum: 26ac9a20c8043a28d373bfe0ca361cc6
     - path: miniwdl_run/wdl/workflows/theiaprok/wf_theiaprok_illumina_se.wdl
-      md5sum: e1d9e75dae5176ceeb95b88a5d3bbba7
+      md5sum: 7303247badeb82119ccef528f7367f89
     - path: miniwdl_run/wdl/workflows/utilities/wf_merlin_magic.wdl
       md5sum: 00bd2489b2a7aa5b88340a940961a857
     - path: miniwdl_run/wdl/workflows/utilities/wf_read_QC_trim_se.wdl

workflows/theiaprok/wf_theiaprok_fasta.wdl

@@ -175,6 +175,7 @@ workflow theiaprok_fasta {
         ani_output_tsv = ani.ani_output_tsv,
         ani_top_species_match = ani.ani_top_species_match,
         ani_mummer_version = ani.ani_mummer_version,
+        ani_docker = ani.ani_docker,
         kmerfinder_docker = kmerfinder.kmerfinder_docker,
         kmerfinder_results_tsv = kmerfinder.kmerfinder_results_tsv,
         kmerfinder_top_hit = kmerfinder.kmerfinder_top_hit,
@@ -427,6 +428,7 @@ workflow theiaprok_fasta {
     File? ani_output_tsv = ani.ani_output_tsv
     String? ani_top_species_match = ani.ani_top_species_match
     String? ani_mummer_version = ani.ani_mummer_version
+    String? ani_mummer_docker = ani.ani_docker
     # kmerfinder outputs
     String? kmerfinder_docker = kmerfinder.kmerfinder_docker
     File? kmerfinder_results_tsv = kmerfinder.kmerfinder_results_tsv

workflows/theiaprok/wf_theiaprok_illumina_pe.wdl

@@ -300,6 +300,7 @@ workflow theiaprok_illumina_pe {
             ani_output_tsv = ani.ani_output_tsv,
             ani_top_species_match = ani.ani_top_species_match,
             ani_mummer_version = ani.ani_mummer_version,
+            ani_docker = ani.ani_docker,
             kmerfinder_docker = kmerfinder.kmerfinder_docker,
             kmerfinder_results_tsv = kmerfinder.kmerfinder_results_tsv,
             kmerfinder_top_hit = kmerfinder.kmerfinder_top_hit,
@@ -621,6 +622,7 @@ workflow theiaprok_illumina_pe {
     File? ani_output_tsv = ani.ani_output_tsv
     String? ani_top_species_match = ani.ani_top_species_match
     String? ani_mummer_version = ani.ani_mummer_version
+    String? ani_mummer_docker = ani.ani_docker
     # kmerfinder outputs
     String? kmerfinder_docker = kmerfinder.kmerfinder_docker
     File? kmerfinder_results_tsv = kmerfinder.kmerfinder_results_tsv

workflows/theiaprok/wf_theiaprok_illumina_se.wdl

@@ -273,6 +273,7 @@ workflow theiaprok_illumina_se {
             ani_output_tsv = ani.ani_output_tsv,
             ani_top_species_match = ani.ani_top_species_match,
             ani_mummer_version = ani.ani_mummer_version,
+            ani_docker = ani.ani_docker,
             kmerfinder_docker = kmerfinder.kmerfinder_docker,
             kmerfinder_results_tsv = kmerfinder.kmerfinder_results_tsv,
             kmerfinder_top_hit = kmerfinder.kmerfinder_top_hit,
@@ -573,6 +574,7 @@ workflow theiaprok_illumina_se {
     File? ani_output_tsv = ani.ani_output_tsv
     String? ani_top_species_match = ani.ani_top_species_match
     String? ani_mummer_version = ani.ani_mummer_version
+    String? ani_mummer_docker = ani.ani_docker
     # kmerfinder outputs
     String? kmerfinder_docker = kmerfinder.kmerfinder_docker
     File? kmerfinder_results_tsv = kmerfinder.kmerfinder_results_tsv

workflows/theiaprok/wf_theiaprok_ont.wdl

@@ -271,6 +271,7 @@ workflow theiaprok_ont {
             ani_output_tsv = ani.ani_output_tsv,
             ani_top_species_match = ani.ani_top_species_match,
             ani_mummer_version = ani.ani_mummer_version,
+            ani_docker = ani.ani_docker,
             kmerfinder_docker = kmerfinder.kmerfinder_docker,
             kmerfinder_results_tsv = kmerfinder.kmerfinder_results_tsv,
             kmerfinder_top_hit = kmerfinder.kmerfinder_top_hit,
@@ -547,6 +548,7 @@ workflow theiaprok_ont {
     File? ani_output_tsv = ani.ani_output_tsv
     String? ani_top_species_match = ani.ani_top_species_match
     String? ani_mummer_version = ani.ani_mummer_version
+    String? ani_mummer_docker = ani.ani_docker
     # kmerfinder outputs
     String? kmerfinder_docker = kmerfinder.kmerfinder_docker
     File? kmerfinder_results_tsv = kmerfinder.kmerfinder_results_tsv