diff --git a/README.md b/README.md
index 1a4fe4392..03d48ce9f 100644
--- a/README.md
+++ b/README.md
@@ -66,10 +66,16 @@ seqtk sample -s 42 SRR6109255_1.fastq.gz 0.01 | gzip -c > tiny/SRR6109255_1.fast
 seqtk sample -s 42 SRR6109255_2.fastq.gz 0.01 | gzip -c > tiny/SRR6109255_2.fastq.gz
 ```
 
+A FASTQ of only the UMI sequences was generated by slicing the first 10bp from the sequencing data in the R1 FASTQ file using fastx:
 
-|    tool |  version |
-|---------|----------|
-| `seqtk` | 1.4-r122 |
+```console
+gunzip -dc testdata/duplex-seq/tiny/SRR6109255_1.fastq.gz | fastx_trimmer -f 1 -l 10 -z -o testdata/duplex-seq/tiny/SRR6109255_3.fastq.gz
+```
+
+|    tool       |  version |
+|---------------|----------|
+| fastx_toolkit | 0.0.14   |
+| `seqtk`       | 1.4-r122 |
 
 ### Reference Information
 
diff --git a/testdata/duplex-seq/tiny/SRR6109255_3.fastq.gz b/testdata/duplex-seq/tiny/SRR6109255_3.fastq.gz
new file mode 100644
index 000000000..c39ac40dd
Binary files /dev/null and b/testdata/duplex-seq/tiny/SRR6109255_3.fastq.gz differ
diff --git a/testdata/duplex-seq/tiny/samplesheet.csv b/testdata/duplex-seq/tiny/samplesheet.csv
index 71a291cce..79d27abb5 100644
--- a/testdata/duplex-seq/tiny/samplesheet.csv
+++ b/testdata/duplex-seq/tiny/samplesheet.csv
@@ -1,2 +1,3 @@
-sample,id,single_end,fastq_1,fastq_2,read_structure
-SRR6109255,SRR6109255,false,https://raw.githubusercontent.com/nf-core/test-datasets/fastquorum/testdata/duplex-seq/tiny/SRR6109255_1.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/fastquorum/testdata/duplex-seq/tiny/SRR6109255_2.fastq.gz,10M1S+T 10M1S+T
+sample,id,single_end,fastq_1,fastq_2,fastq_3,read_structure
+SRR6109255,SRR6109255,false,https://raw.githubusercontent.com/nf-core/test-datasets/fastquorum/testdata/duplex-seq/tiny/SRR6109255_1.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/fastquorum/testdata/duplex-seq/tiny/SRR6109255_2.fastq.gz,,10M1S+T 10M1S+T
+SRR6109255_2,SRR6109255_2,false,https://raw.githubusercontent.com/nf-core/test-datasets/fastquorum/testdata/duplex-seq/tiny/SRR6109255_1.fastq.gz,https://raw.githubusercontent.com/nf-core/test-datasets/fastquorum/testdata/duplex-seq/tiny/SRR6109255_2.fastq.gz,https://github.com/nf-core/test-datasets/raw/fastquorum_umi_fastq_file/testdata/duplex-seq/tiny/SRR6109255_3.fastq.gz,11S+T 11S+T +M