diff --git a/tools/rasusa/.lint_skip b/tools/rasusa/.lint_skip
deleted file mode 100644
index 2092d1d0d68..00000000000
--- a/tools/rasusa/.lint_skip
+++ /dev/null
@@ -1 +0,0 @@
-TestsCaseValidation
diff --git a/tools/rasusa/rasusa.xml b/tools/rasusa/rasusa.xml
index 87dddb954d3..642f430ebb2 100644
--- a/tools/rasusa/rasusa.xml
+++ b/tools/rasusa/rasusa.xml
@@ -1,8 +1,9 @@
-<tool id="rasusa" name="rasusa" version="@TOOL_VERSION@+galaxy@VERSION_SUFFIX@" profile="22.05">
+<tool id="rasusa" name="rasusa" version="@TOOL_VERSION@+galaxy@VERSION_SUFFIX@" profile="@PROFILE@">
     <description>Randomly subsample reads to a specified coverage</description>
     <macros>
-        <token name="@TOOL_VERSION@">3.0.0</token>
+        <token name="@TOOL_VERSION@">4.0.0</token>
         <token name="@VERSION_SUFFIX@">0</token>
+        <token name="@PROFILE@">25.0</token>
         <token name="@FORMATS@">fastqsanger,fastqsanger.gz,fasta,fasta.gz</token>
         <xml name="size_units">
             <option value="b">bases</option>
@@ -52,7 +53,7 @@
 --frac $subsample.frac
 #end if
 #if $r1_ext.endswith(".gz") or $r2_ext.endswith(".gz")
---output-type g
+--compress-type g
 #end if    ]]>
     </token>
     </macros>
@@ -65,51 +66,53 @@
     </requirements>
 
     <command detect_errors="exit_code"><![CDATA[
-#if str( $input.input_selector ) == "aligned":
-ln -s '$bam' 'input.bam' &&
-ln -s '$bam.metadata.bam_index' 'input.bam.bai' &&
-rasusa aln
---coverage $input.coverage
---step-size $input.step_size
+#if str($input.input_selector) == "aligned":
+    #if '$aligned_input.ext' == "bam":
+        ln -s '$aligned_input.metadata.bam_index' 'input.bam.bai' &&
+    #end if
+    ln -s '$aligned_input' input.$aligned_input.ext &&
+    rasusa aln
+    --coverage $input.coverage
+    --step-size $input.step_size
 #else:
-rasusa reads
+    rasusa reads
 #end if
 
 #if $seed
--s $seed
+    -s $seed
 #end if
 
-#if str( $input.input_selector ) == "paired":
+#if str($input.input_selector) == "paired":
     #set r1_ext = $input.reads1.extension
     #set r2_ext = $input.reads2.extension
--o 'paired_out1.$r1_ext'
--o 'paired_out2.$r2_ext'
-@FASTQ_SUBSAMPLE_OPTIONS@
-'${input.reads1}'
-'${input.reads2}' &&
-mv 'paired_out1.$r1_ext' '$paired_output1' &&
-mv 'paired_out2.$r2_ext' '$paired_output2'
+    -o 'paired_out1.$r1_ext'
+    -o 'paired_out2.$r2_ext'
+    @FASTQ_SUBSAMPLE_OPTIONS@
+    '${input.reads1}'
+    '${input.reads2}' &&
+    mv 'paired_out1.$r1_ext' '$paired_output1' &&
+    mv 'paired_out2.$r2_ext' '$paired_output2'
 
-#elif str( $input.input_selector ) == "paired_collection":
+#elif str($input.input_selector) == "paired_collection":
     #set r1_ext = $input.collection.forward.extension
     #set r2_ext = $input.collection.reverse.extension
--o 'paired_out1.$r1_ext'
--o 'paired_out2.$r2_ext'
-@FASTQ_SUBSAMPLE_OPTIONS@
-'${input.collection.forward}'
-'${input.collection.reverse}' &&
-mv 'paired_out1.$r1_ext' '${collection_output.forward}' &&
-mv 'paired_out2.$r2_ext' '${collection_output.reverse}'
+    -o 'paired_out1.$r1_ext'
+    -o 'paired_out2.$r2_ext'
+    @FASTQ_SUBSAMPLE_OPTIONS@
+    '${input.collection.forward}'
+    '${input.collection.reverse}' &&
+    mv 'paired_out1.$r1_ext' '${collection_output.forward}' &&
+    mv 'paired_out2.$r2_ext' '${collection_output.reverse}'
 
-#elif str( $input.input_selector ) == "single":
+#elif str($input.input_selector) == "single":
     #set r1_ext = $input.reads.extension
--o 'single_out.$r1_ext'
-@FASTQ_SUBSAMPLE_OPTIONS@
-'${input.reads}' &&
-mv 'single_out.$r1_ext' '$single_output'
+    -o 'single_out.$r1_ext'
+    @FASTQ_SUBSAMPLE_OPTIONS@
+    '${input.reads}' &&
+    mv 'single_out.$r1_ext' '$single_output'
 
-#elif str( $input.input_selector ) == "aligned":
-'input.bam' | samtools sort --no-PG -@ 1 -T '\${TMPDIR:-.}' -O bam -o '$bam_output' -
+#elif str($input.input_selector) == "aligned":
+    --output-format bam 'input.$aligned_input.ext' | samtools sort --no-PG -@ 1 -T '\${TMPDIR:-.}' -O bam -o '$bam_output' -
 #end if
     ]]></command>
     <inputs>
@@ -134,7 +137,7 @@ mv 'single_out.$r1_ext' '$single_output'
                 <expand macro="params_fastq" />
             </when>
             <when value="aligned">
-                <param name="bam" format="sam,bam" type="data" label="Select BAM file(s) with alignments"/>
+                <param name="aligned_input" format="bam" type="data" label="Select BAM file with alignments"/>
                 <param argument="--coverage" type="integer" min="0" optional="true" value="" label="The desired depth of coverage to subsample the alignment to"/>
                 <param type="integer" argument="--step-size" value="100" label="When a region has less than the desired coverage, the step size to move along the chromosome to find more reads."
                     help="The lowest of the step and the minimum end coordinate of the reads in the region will be used. This parameter can have a significant impact on the runtime of the subsampling process."/>
@@ -167,12 +170,12 @@ mv 'single_out.$r1_ext' '$single_output'
             <conditional name="input">
                 <param name="input_selector" value="single"/>
                 <param name="reads" value="r1.fastq.gz"/>
-            </conditional>
-            <conditional name="subsample">
-                <param name="type" value="coverage"/>
-                <param name="genome_size_unit" value="b"/>
-                <param name="genome_size" value="1000"/>
-                <param name="coverage" value="1"/>
+                <conditional name="subsample">
+                    <param name="type" value="coverage"/>
+                    <param name="genome_size_unit" value="b"/>
+                    <param name="genome_size" value="1000"/>
+                    <param name="coverage" value="1"/>
+                </conditional>
             </conditional>
             <param name="seed" value="1"/>
             <output name="single_output" value="single_by_coverage_b.fastq.gz" ftype="fastqsanger.gz"/>
@@ -183,12 +186,12 @@ mv 'single_out.$r1_ext' '$single_output'
                 <param name="input_selector" value="paired"/>
                 <param name="reads1" value="r1.fastq.gz"/>
                 <param name="reads2" value="r2.fastq.gz"/>
-            </conditional>
-            <conditional name="subsample">
-                <param name="type" value="coverage"/>
-                <param name="genome_size_unit" value="k"/>
-                <param name="genome_size" value="1"/>
-                <param name="coverage" value="1"/>
+                <conditional name="subsample">
+                    <param name="type" value="coverage"/>
+                    <param name="genome_size_unit" value="k"/>
+                    <param name="genome_size" value="1"/>
+                    <param name="coverage" value="1"/>
+                </conditional>
             </conditional>
             <param name="seed" value="1"/>
             <output name="paired_output1" value="paired1_by_coverage_k.fastq.gz" ftype="fastqsanger.gz"/>
@@ -204,12 +207,12 @@ mv 'single_out.$r1_ext' '$single_output'
                         <element name="reverse" value="r2.fastq.gz"/>
                     </collection>
                 </param>
-            </conditional>
-            <conditional name="subsample">
-                <param name="type" value="coverage"/>
-                <param name="genome_size_unit" value="m"/>
-                <param name="genome_size" value="0.001"/>
-                <param name="coverage" value="1"/>
+                <conditional name="subsample">
+                    <param name="type" value="coverage"/>
+                    <param name="genome_size_unit" value="m"/>
+                    <param name="genome_size" value="0.001"/>
+                    <param name="coverage" value="1"/>
+                </conditional>
             </conditional>
             <param name="seed" value="1"/>
             <output_collection name="collection_output" type="paired">
@@ -222,12 +225,12 @@ mv 'single_out.$r1_ext' '$single_output'
             <conditional name="input">
                 <param name="input_selector" value="single"/>
                 <param name="reads" value="r1.fasta.gz"/>
-            </conditional>
-            <conditional name="subsample">
-                <param name="type" value="coverage"/>
-                <param name="genome_size_unit" value="g"/>
-                <param name="genome_size" value="0.001"/>
-                <param name="coverage" value="0.001"/>
+                <conditional name="subsample">
+                    <param name="type" value="coverage"/>
+                    <param name="genome_size_unit" value="g"/>
+                    <param name="genome_size" value="0.001"/>
+                    <param name="coverage" value="0.001"/>
+                </conditional>
             </conditional>
             <param name="seed" value="1"/>
             <output name="single_output" value="single_end_by_coverage_g.fasta" ftype="fasta.gz"/>
@@ -238,11 +241,11 @@ mv 'single_out.$r1_ext' '$single_output'
                 <param name="input_selector" value="paired"/>
                 <param name="reads1" value="r1.fastq"/>
                 <param name="reads2" value="r2.fastq"/>
-            </conditional>
-            <conditional name="subsample">
-                <param name="type" value="num_bases"/>
-                <param name="num_bases_unit" value="k"/>
-                <param name="bases" value="2"/>
+                <conditional name="subsample">
+                    <param name="type" value="num_bases"/>
+                    <param name="num_bases_unit" value="k"/>
+                    <param name="bases" value="2"/>
+                </conditional>
             </conditional>
             <param name="seed" value="1"/>
             <output name="paired_output1" value="paired1_by_num_bases_k.fastq" ftype="fastqsanger"/>
@@ -254,10 +257,10 @@ mv 'single_out.$r1_ext' '$single_output'
                 <param name="input_selector" value="paired"/>
                 <param name="reads1" value="r1.fasta.gz"/>
                 <param name="reads2" value="r2.fasta.gz"/>
-            </conditional>
-            <conditional name="subsample">
-                <param name="type" value="num_reads"/>
-                <param name="num" value="5"/>
+                <conditional name="subsample">
+                    <param name="type" value="num_reads"/>
+                    <param name="num" value="5"/>
+                </conditional>
             </conditional>
             <param name="seed" value="1"/>
             <output name="paired_output1" value="paired1_by_num_reads.fasta.gz" ftype="fasta.gz"/>
@@ -273,10 +276,10 @@ mv 'single_out.$r1_ext' '$single_output'
                         <element name="reverse" value="r2.fasta"/>
                     </collection>
                 </param>
-            </conditional>
-            <conditional name="subsample">
-                <param name="type" value="frac_reads"/>
-                <param name="frac" value="0.6"/>
+                <conditional name="subsample">
+                    <param name="type" value="frac_reads"/>
+                    <param name="frac" value="0.6"/>
+                </conditional>
             </conditional>
             <param name="seed" value="1"/>
             <output_collection name="collection_output" type="paired">
@@ -285,12 +288,12 @@ mv 'single_out.$r1_ext' '$single_output'
             </output_collection>
         </test>
         <test expect_num_outputs="1">
-            <!-- test 8: bam input -->
+            <!-- test 8: bam input, bam output -->
             <conditional name="input">
                 <param name="input_selector" value="aligned"/>
-                <param name="bam" value="input.bam" />
+                <param name="aligned_input" value="input.bam" />
+                <param name="coverage" value="1"/>
             </conditional>
-            <param name="coverage" value="1"/>
             <param name="seed" value="1"/>
             <output name="bam_output" value="output.bam" ftype="bam"/>
         </test>
@@ -306,4 +309,4 @@ specifying the size of the subset:
     <citations>
         <citation type="doi">10.21105/joss.03941</citation>
     </citations>
-</tool>
+</tool>
\ No newline at end of file
diff --git a/tools/rasusa/test-data/output.bam b/tools/rasusa/test-data/output.bam
index 6be07c5ccb5..bc86151512b 100644
Binary files a/tools/rasusa/test-data/output.bam and b/tools/rasusa/test-data/output.bam differ