# Lost majority of the reads after kneadata **URL:** \ **Category:** KneadData\ **Created:** [December 3, 2022, 6:25pm UTC](https://forum.biobakery.org/t/lost-majority-of-the-reads-after-kneadata/4477 "2022-12-03T18:25:55Z")\ **Posts on this page:** 2\ **Page:** 1
**Author:** ![LuZhang](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/luzhang/32/623_2.png) [@LuZhang](https://forum.biobakery.org/u/LuZhang)\ **Post date:** [December 3, 2022, 6:25pm UTC](https://forum.biobakery.org/t/lost-majority-of-the-reads-after-kneadata/4477/1 "2022-12-03T18:25:56Z")
Hi 🙂 When I use Kneaddata (v0.12.0) to trim sequences files and separate the reads from host(mouse), very few reads are left after the whole pipeline. I am using the default parameters. Command: `kneaddata --input1 sample1.fq --input2 sample2.fq -db GRCm39_mice_db --output /output/ -t 20 --run-fastqc-start --run-fastqc-end --trimmomatic /home/software/trimmomatic/Trimmomatic-0.39 --sequencer-source none` one example of the log file: ```auto 11/30/2022 05:52:16 PM - kneaddata.utilities - INFO: Running bowtie2 ... 11/30/2022 05:52:16 PM - kneaddata.utilities - INFO: Execute command: kneaddata_bowtie2_discordant_pairs --bowtie2 /home/lzhang/miniconda3/envs/mice_project/bin/bowtie2 --threads 20 -x /sbidata/projects/lzhang/2022_mice/Analysis/kneaddata_database/GRCm39/GRCm39_mice_db --mode strict --bowtie2-options "--very-sensitive-local --phred33" -1 /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata.repeats.removed.1.fastq -2 /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata.repeats.removed.2.fastq --un-pair /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_paired_clean_%.fastq --al-pair /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_paired_contam_%.fastq -U /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata.repeats.removed.unmatched.1.fastq,/sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata.repeats.removed.unmatched.2.fastq --un-single /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_unmatched_%_clean.fastq --al-single /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_unmatched_%_contam.fastq -S /dev/null 11/30/2022 06:01:17 PM - kneaddata.utilities - DEBUG: b'6279552 reads; of these:\n 6279552 (100.00%) were unpaired; of these:\n 6541 (0.10%) aligned 0 times\n 930147 (14.81%) aligned exactly 1 time\n 5342864 (85.08%) aligned >1 times\n99.90% overall alignment rate\npair1_aligned : 2691082\npair2_aligned : 2691082\npair1_unaligned : 2140\npair2_unaligned : 2140\norphan1_aligned : 434424\norphan2_aligned : 457777\norphan1_unaligned : 406\norphan2_unaligned : 501\n' 11/30/2022 06:01:17 PM - kneaddata.utilities - DEBUG: Checking output file from bowtie2 : /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_paired_clean_1.fastq 11/30/2022 06:01:17 PM - kneaddata.utilities - DEBUG: Checking output file from bowtie2 : /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_paired_clean_2.fastq 11/30/2022 06:01:18 PM - kneaddata.run - INFO: Total contaminate sequences in file ( /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_paired_contam_1.fastq ) : 2691082.0 11/30/2022 06:01:20 PM - kneaddata.run - INFO: Total contaminate sequences in file ( /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_paired_contam_2.fastq ) : 2691082.0 11/30/2022 06:01:20 PM - kneaddata.run - INFO: Total contaminate sequences in file ( /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_unmatched_1_contam.fastq ) : 434424.0 11/30/2022 06:01:20 PM - kneaddata.run - INFO: Total contaminate sequences in file ( /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_unmatched_2_contam.fastq ) : 457777.0 11/30/2022 06:01:20 PM - kneaddata.utilities - INFO: READ COUNT: decontaminated GRCm39_mice_db pair1 : Total reads after removing those found in reference database ( /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_paired_clean_1.fastq ): 2140.0 11/30/2022 06:01:20 PM - kneaddata.utilities - INFO: READ COUNT: decontaminated GRCm39_mice_db pair2 : Total reads after removing those found in reference database ( /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_GRCm39_mice_db_bowtie2_paired_clean_2.fastq ): 2140.0 11/30/2022 06:01:20 PM - kneaddata.utilities - INFO: READ COUNT: final pair1 : Total reads after merging results from multiple databases ( /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_paired_1.fastq ): 2140.0 11/30/2022 06:01:20 PM - kneaddata.utilities - INFO: READ COUNT: final pair2 : Total reads after merging results from multiple databases ( /sbidata/projects/lzhang/2022_mice/Data/rawData_part2/processed/D14_4F8_1.new_kneaddata_paired_2.fastq ): 2140.0 ``` overview of the whole procedure count number | Sample | raw pair1 | raw pair2 | trimmed pair1 | trimmed pair2 | trimmed orphan1 | trimmed orphan2 | decontaminated GRCm39\_mice\_db pair1 | decontaminated GRCm39\_mice\_db pair2 | decontaminated GRCm39\_mice\_db orphan1 | decontaminated GRCm39\_mice\_db orphan2 | final pair1 | final pair2 | final orphan1 | final orphan2 | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | my\_sample | 3872223 | 3872223 | 3489545 | 3489545 | 164393 | 157032 | 2140 | 2140 | 406 | 501 | 2140 | 2140 | 406 | 501 | Some samples have relatively okay results, but some have few reads left. I am not sure whether is too much contaminated or something went wrong in the bowtie procedure? Thanks a lot! ---
**Author:** ![adamsorbie](https://yyz1.discourse-cdn.com/flex027/user_avatar/forum.biobakery.org/adamsorbie/32/2099_2.png) [@adamsorbie](https://forum.biobakery.org/u/adamsorbie)\ **Post date:** [May 24, 2023, 4:46pm UTC](https://forum.biobakery.org/t/lost-majority-of-the-reads-after-kneadata/4477/2 "2023-05-24T16:46:59Z")
Hey, I have a similar problem, did you manage to figure out how to solve this?