The error output of Kneaddata when demo.fastq was used?

[Ming Liao]

I am running kneaddata with the "demo.fastq" in the examples of "HUMAnN2". It came up with the following error. 

What is the right output of it? 

liaoming@biolinux~/kneaddata_v0.5.1/db

# kneaddata --input ../../humann2_v0.6.0/examples/demo.fastq --reference-db ./Homo_sapiens_Bowtie2_v0.1 --output kneaddata_demo_output

Initial number of reads ( /home/liaoming/humann2_v0.6.0/examples/demo.fastq ): 12500

Running Trimmomatic ... 

ERROR: Trimmomatic created empty output files.

Then I am looking into the log file:

liaoming@biolinux~/kneaddata_v0.5.1/db/kneaddata_demo_output

# less demo_kneaddata.log 

02/11/2016 06:34:10 PM - kneaddata.knead_data - INFO: Running kneaddata v0.5.1

02/11/2016 06:34:10 PM - kneaddata.knead_data - INFO: Output files will be written to: /home/liaoming/kneaddata_v0.5.1/db/kneaddata_demo_output

02/11/2016 06:34:10 PM - kneaddata.knead_data - DEBUG: Running with the following arguments: 

verbose = False

bmtagger_path = None

minscore = 50

bowtie2_path = /appl/bin/bowtie2

maxperiod = 500

fastqc_start = False

bmtagger = False

log_level = DEBUG

log = /home/liaoming/kneaddata_v0.5.1/db/kneaddata_demo_output/demo_kneaddata.log

max_memory = 500m

fastqc_path = None

output_dir = /home/liaoming/kneaddata_v0.5.1/db/kneaddata_demo_output

trf_path = None

reference_db = /home/liaoming/kneaddata_v0.5.1/db/Homo_sapiens_Bowtie2_v0.1/Homo_sapiens

input = /home/liaoming/humann2_v0.6.0/examples/demo.fastq

pi = 10

match = 2

pm = 80

trimmomatic_path = /home/liaoming/tools/Trimmomatic-0.33/trimmomatic-0.33.jar

store_temp_output = False

mismatch = 7

mismatch = 7

threads = 1

delta = 7

bowtie2_options = --very-sensitive --phred33

bypass_trim = False

processes = 1

trimmomatic_quality_scores = -phred33

fastqc_end = False

trf = False

trimmomatic_options = None

output_prefix = demo_kneaddata

remove_temp_output = True

02/11/2016 06:34:10 PM - kneaddata.utilities - INFO: Initial number of reads ( /home/liaoming/humann2_v0.6.0/examples/demo.fastq ): 12500

02/11/2016 06:34:10 PM - kneaddata.utilities - DEBUG: Checking input file to Trimmomatic : /home/liaoming/humann2_v0.6.0/examples/demo.fastq

02/11/2016 06:34:10 PM - kneaddata.utilities - INFO: Running Trimmomatic ... 

02/11/2016 06:34:10 PM - kneaddata.utilities - INFO: Execute command: java -Xmx500m -d64 -jar /home/liaoming/tools/Trimmomatic-0.33/trimmomatic-0.33.jar SE -threads 1 -phred33 /home/liaoming/humann2_v0.6.0/examples/demo.fastq /home/liaoming/kneaddata_v0.5.1/db/kneaddata_demo_output/demo_kneaddata.trimmed.fastq SLIDINGWINDOW:4:20 MINLEN:105

02/11/2016 06:34:11 PM - kneaddata.utilities - DEBUG: TrimmomaticSE: Started with arguments: -threads 1 -phred33 /home/liaoming/humann2_v0.6.0/examples/demo.fastq /home/liaoming/kneaddata_v0.5.1/db/kneaddata_demo_output/demo_kneaddata.trimmed.fastq SLIDINGWINDOW:4:20 MINLEN:105

Input Reads: 12500 Surviving: 0 (0.00%) Dropped: 12500 (100.00%)

TrimmomaticSE: Completed successfully

02/11/2016 06:34:11 PM - kneaddata.utilities - DEBUG: Checking output file from Trimmomatic : /home/liaoming/knead

[Lauren McIver]

Hi Ming,

Yes, this is expected. When using the humann2 demo.fastq file as input to kneaddata, all reads are filtered by trimmomatic because the quality scores do not meet the cutoff from the kneaddata trimmomatic default settings (ie "SLIDINGWINDOW:4:20"). If you try the run again, decreasing the sliding window quality score value from 20 to 15 you should see trimmomatic not filter any sequences and the run will finish without error.

Also if you would like to try another demo there is one for kneaddata located in the folder kneaddata/examples/demo.fastq. When running this demo input file, you should see trimmomatic process the file and then 10 reads will be removed after the bowtie2 comparison to the reference database. 

Thank you,

Lauren

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