I run kneaddata(v0.10.0) , it output empty files, but with no error information.
The log file as follow:
12/10/2021 04:46:24 PM - kneaddata.knead_data - INFO: Running kneaddata v0.10.0 12/10/2021 04:46:24 PM - kneaddata.knead_data - INFO: Output files will be written to: /public4/home/sc56690/data/temp/qc 12/10/2021 04:46:24 PM - kneaddata.knead_data - DEBUG: Running with the following arguments: verbose = True bypass_trf = True bmtagger_path = None minscore = 50 bowtie2_path = /public4/home/sc56690/.conda/envs/humann2/bin/bowtie2 maxperiod = 500 discordant = True serial = False fastqc_start = False store_temp_output = False cat_final_output = False log_level = DEBUG log = /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.log sequencer_source = NexteraPE max_memory = 500m remove_intermediate_output = True fastqc_path = None output_dir = /public4/home/sc56690/data/temp/qc trf_path = None remove_temp_output = True reference_db = /public4/home/sc56690/db/kneaddata/human_genome/Homo_sapiens input = /public4/home/sc56690/data/seq/1-1106_FDSW202359622-1r_1.fq /public4/home/sc56690/data/seq/1-1106_FDSW202359622-1r_2.fq decontaminate_pairs = strict reorder = False pm = 80 trimmomatic_path = /public4/home/sc56690/.conda/envs/humann2/share/trimmomatic/trimmomatic.jar run_trf = False mismatch = 7 threads = 60 delta = 7 bowtie2_options = --very-sensitive --dovetail --phred33 bypass_trim = False processes = 1 pi = 10 trimmomatic_quality_scores = -phred33 fastqc_end = False trimmomatic_options = ILLUMINACLIP:/public4/home/sc56690/.conda/envs/humann2/share/trimmomatic/adapters/TruSeq2-PE.fa:2:40:15 SLIDINGWINDOW:4:20 MINLEN:50 output_prefix = 1-1106_FDSW202359622-1r_1_kneaddata match = 2 bmtagger = False run_trim_repetitive = False
12/10/2021 04:46:24 PM - kneaddata.utilities - INFO: Reformatting file sequence identifiers ... 12/10/2021 04:47:48 PM - kneaddata.utilities - INFO: Reformatting file sequence identifiers ... 12/10/2021 04:49:14 PM - kneaddata.utilities - INFO: Reordering read identifiers ... 12/10/2021 05:09:34 PM - kneaddata.utilities - INFO: READ COUNT: raw pair1 : Initial number of reads ( /public4/home/sc56690/data/temp/qc/reordered_QejgrJ_reformatted_identifiersxZBJaz_1-1106_FDSW202359622-1r_1 ): 36029417 12/10/2021 05:09:46 PM - kneaddata.utilities - INFO: READ COUNT: raw pair2 : Initial number of reads ( /public4/home/sc56690/data/temp/qc/reordered_EY7eUu_reformatted_identifiersL3KgSn_1-1106_FDSW202359622-1r_2 ): 36029417 12/10/2021 05:09:46 PM - kneaddata.utilities - DEBUG: Checking input file to Trimmomatic : /public4/home/sc56690/data/temp/qc/reordered_QejgrJ_reformatted_identifiersxZBJaz_1-1106_FDSW202359622-1r_1 12/10/2021 05:09:46 PM - kneaddata.utilities - DEBUG: Checking input file to Trimmomatic : /public4/home/sc56690/data/temp/qc/reordered_EY7eUu_reformatted_identifiersL3KgSn_1-1106_FDSW202359622-1r_2 12/10/2021 05:09:46 PM - kneaddata.utilities - INFO: Running Trimmomatic ... 12/10/2021 05:09:46 PM - kneaddata.utilities - INFO: Execute command: java -Xmx500m -jar /public4/home/sc56690/.conda/envs/humann2/share/trimmomatic/trimmomatic.jar PE -threads 60 -phred33 /public4/home/sc56690/data/temp/qc/reordered_QejgrJ_reformatted_identifiersxZBJaz_1-1106_FDSW202359622-1r_1 /public4/home/sc56690/data/temp/qc/reordered_EY7eUu_reformatted_identifiersL3KgSn_1-1106_FDSW202359622-1r_2 /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.1.fastq /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.1.fastq /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.2.fastq /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.2.fastq ILLUMINACLIP:/public4/home/sc56690/.conda/envs/humann2/share/trimmomatic/adapters/TruSeq2-PE.fa:2:40:15 SLIDINGWINDOW:4:20 MINLEN:50 12/10/2021 05:12:07 PM - kneaddata.utilities - DEBUG: TrimmomaticPE: Started with arguments: -threads 60 -phred33 /public4/home/sc56690/data/temp/qc/reordered_QejgrJ_reformatted_identifiersxZBJaz_1-1106_FDSW202359622-1r_1 /public4/home/sc56690/data/temp/qc/reordered_EY7eUu_reformatted_identifiersL3KgSn_1-1106_FDSW202359622-1r_2 /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.1.fastq /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.1.fastq /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.2.fastq /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.2.fastq ILLUMINACLIP:/public4/home/sc56690/.conda/envs/humann2/share/trimmomatic/adapters/TruSeq2-PE.fa:2:40:15 SLIDINGWINDOW:4:20 MINLEN:50 Using PrefixPair: 'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT' and 'CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT' Using Long Clipping Sequence: 'AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT' Using Long Clipping Sequence: 'AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCGATCTCGTATGCCGTCTTCTGCTTG' Using Long Clipping Sequence: 'TTTTTTTTTTAATGATACGGCGACCACCGAGATCTACAC' Using Long Clipping Sequence: 'TTTTTTTTTTCAAGCAGAAGACGGCATACGA' Using Long Clipping Sequence: 'CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT' Using Long Clipping Sequence: 'AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT' ILLUMINACLIP: Using 1 prefix pairs, 6 forward/reverse sequences, 0 forward only sequences, 0 reverse only sequences Input Read Pairs: 36029417 Both Surviving: 34592816 (96.01%) Forward Only Surviving: 527242 (1.46%) Reverse Only Surviving: 603448 (1.67%) Dropped: 305911 (0.85%) TrimmomaticPE: Completed successfully
12/10/2021 05:12:07 PM - kneaddata.utilities - DEBUG: Checking output file from Trimmomatic : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.1.fastq 12/10/2021 05:12:07 PM - kneaddata.utilities - DEBUG: Checking output file from Trimmomatic : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.1.fastq 12/10/2021 05:12:07 PM - kneaddata.utilities - DEBUG: Checking output file from Trimmomatic : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.2.fastq 12/10/2021 05:12:07 PM - kneaddata.utilities - DEBUG: Checking output file from Trimmomatic : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.2.fastq 12/10/2021 05:12:20 PM - kneaddata.utilities - INFO: READ COUNT: trimmed pair1 : Total reads after trimming ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.1.fastq ): 34592816 12/10/2021 05:12:34 PM - kneaddata.utilities - INFO: READ COUNT: trimmed pair2 : Total reads after trimming ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.2.fastq ): 34592816 12/10/2021 05:12:34 PM - kneaddata.utilities - INFO: READ COUNT: trimmed orphan1 : Total reads after trimming ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.1.fastq ): 527242 12/10/2021 05:12:34 PM - kneaddata.utilities - INFO: READ COUNT: trimmed orphan2 : Total reads after trimming ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.2.fastq ): 603448 12/10/2021 05:12:34 PM - kneaddata.run - INFO: Decontaminating ... 12/10/2021 05:12:35 PM - kneaddata.utilities - DEBUG: Checking input file to bowtie2 : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.1.fastq 12/10/2021 05:12:35 PM - kneaddata.utilities - DEBUG: Checking input file to bowtie2 : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.2.fastq 12/10/2021 05:12:35 PM - kneaddata.utilities - DEBUG: Checking input file to bowtie2 : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.1.fastq 12/10/2021 05:12:35 PM - kneaddata.utilities - DEBUG: Checking input file to bowtie2 : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.2.fastq 12/10/2021 05:12:35 PM - kneaddata.utilities - INFO: Running bowtie2 ... 12/10/2021 05:12:35 PM - kneaddata.utilities - INFO: Execute command: kneaddata_bowtie2_discordant_pairs --bowtie2 /public4/home/sc56690/.conda/envs/humann2/bin/bowtie2 --threads 60 -x /public4/home/sc56690/db/kneaddata/human_genome/Homo_sapiens --mode strict --bowtie2-options "--very-sensitive --dovetail --phred33" -1 /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.1.fastq -2 /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.2.fastq --un-pair /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_clean_%.fastq --al-pair /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_contam_%.fastq -U /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.1.fastq,/public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata.trimmed.single.2.fastq --un-single /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_unmatched_%_clean.fastq --al-single /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_unmatched_%_contam.fastq -S /dev/null 12/10/2021 05:29:20 PM - kneaddata.utilities - DEBUG: 70316322 reads; of these: 70316322 (100.00%) were unpaired; of these: 70313909 (100.00%) aligned 0 times 579 (0.00%) aligned exactly 1 time 1834 (0.00%) aligned >1 times 0.00% overall alignment rate pair1_aligned : 0 pair2_aligned : 0 orphan1_unaligned : 35119566 orphan2_unaligned : 35194343 orphan2_aligned : 1921 pair2_unaligned : 0 pair1_unaligned : 0 orphan1_aligned : 492
12/10/2021 05:29:20 PM - kneaddata.utilities - DEBUG: Checking output file from bowtie2 : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_clean_1.fastq 12/10/2021 05:29:20 PM - kneaddata.utilities - DEBUG: Checking output file from bowtie2 : /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_clean_2.fastq 12/10/2021 05:29:21 PM - kneaddata.run - INFO: Total contaminate sequences in file ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_contam_1.fastq ) : 0 12/10/2021 05:29:21 PM - kneaddata.run - INFO: Total contaminate sequences in file ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_contam_2.fastq ) : 0 12/10/2021 05:29:21 PM - kneaddata.run - INFO: Total contaminate sequences in file ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_unmatched_1_contam.fastq ) : 492 12/10/2021 05:29:21 PM - kneaddata.run - INFO: Total contaminate sequences in file ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_unmatched_2_contam.fastq ) : 1921 12/10/2021 05:29:21 PM - kneaddata.utilities - INFO: READ COUNT: decontaminated Homo_sapiens pair1 : Total reads after removing those found in reference database ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_clean_1.fastq ): 0 12/10/2021 05:29:21 PM - kneaddata.utilities - INFO: READ COUNT: decontaminated Homo_sapiens pair2 : Total reads after removing those found in reference database ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_clean_2.fastq ): 0 12/10/2021 05:29:21 PM - kneaddata.utilities - INFO: READ COUNT: final pair1 : Total reads after merging results from multiple databases ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_paired_1.fastq ): 0 12/10/2021 05:29:21 PM - kneaddata.utilities - INFO: READ COUNT: final pair2 : Total reads after merging results from multiple databases ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_paired_2.fastq ): 0 12/10/2021 05:29:21 PM - kneaddata.utilities - WARNING: Unable to remove file: /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_clean_1.fastq 12/10/2021 05:29:21 PM - kneaddata.utilities - WARNING: Unable to remove file: /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_paired_clean_2.fastq 12/10/2021 05:29:33 PM - kneaddata.utilities - INFO: READ COUNT: decontaminated Homo_sapiens orphan1 : Total reads after removing those found in reference database ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_unmatched_1_clean.fastq ): 35119566 12/10/2021 05:29:46 PM - kneaddata.utilities - INFO: READ COUNT: final orphan1 : Total reads after merging results from multiple databases ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_unmatched_1.fastq ): 35119566 12/10/2021 05:29:46 PM - kneaddata.utilities - WARNING: Unable to remove file: /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_unmatched_1_clean.fastq 12/10/2021 05:30:02 PM - kneaddata.utilities - INFO: READ COUNT: decontaminated Homo_sapiens orphan2 : Total reads after removing those found in reference database ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_unmatched_2_clean.fastq ): 35194343 12/10/2021 05:30:14 PM - kneaddata.utilities - INFO: READ COUNT: final orphan2 : Total reads after merging results from multiple databases ( /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_unmatched_2.fastq ): 35194343 12/10/2021 05:30:14 PM - kneaddata.utilities - WARNING: Unable to remove file: /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_Homo_sapiens_bowtie2_unmatched_2_clean.fastq 12/10/2021 05:30:24 PM - kneaddata.knead_data - INFO: Final output files created: /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_paired_1.fastq /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_paired_2.fastq /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_unmatched_1.fastq /public4/home/sc56690/data/temp/qc/1-1106_FDSW202359622-1r_1_kneaddata_unmatched_2.fastq
I have no idea where is the error, Is there something wrong with "--mode strict" which I not add, but kneaddata set automatically?
Check the input after alignment.
0316322 reads; of these: 70316322 (100.00%) were unpaired; of these: 70313909 (100.00%) aligned 0 times 579 (0.00%) aligned exactly 1 time 1834 (0.00%) aligned >1 times 0.00% overall alignment rate pair1_aligned : 0 pair2_aligned : 0 orphan1_unaligned : 35119566 orphan2_unaligned : 35194343 orphan2_aligned : 1921 pair2_unaligned : 0 pair1_unaligned : 0 orphan1_aligned : 492
I checked the intermediate-output:
and run bowtie2 with command:
and output only three files:
the log information: