cutadapt demux-paired demuxes some but not all samples

mchimenti · October 17, 2020, 5:36pm

Hello:
I am attempting to demultiplex paired-end, 300 bp reads from a MiSeq run with dual-indexed data. Barcodes are in the reads. I checked with 'grep' and they are the first 8 nt in both R1 and R2 reads. They are not reverse complimented.

I am running qiime2-2020.8 installed with conda on Mac OS X (10.15.6 Catalina) within an Ananconda installation.

From within the qiime2 environment I am calling:

qiime cutadapt demux-paired --i-seqs testdata.qza --m-forward-barcodes-file sample_metadata_gvdh.txt --m-forward-barcodes-column BarcodeForward --m-reverse-barcodes-file sample_metadata_gvdh.txt --m-reverse-barcodes-column BarcodeReverse --o-per-sample-sequences testout.qza --o-untrimmed-sequences untrim.qza --verbose

"testdata" here is the first 1M reads from the FASTQ files so that I could test quickly. I have tried on the full dataset (20M reads) and the results are the same.

I have the forward and reverse barcodes in my metadata file, and I can find them in the sequences using grep. They are not reverse-complimented.

When I run qiime cutadapt demux-paired (as shown above), the command completes successfully, but only demuxes a handful of the 36 samples (see below for output in verbose mode).

This appears possibly similar to a bug report from here:

github.com/qiime2/q2-cutadapt

Cutadapt behaving differently inside and outside of qiime2

opened 01:27PM - 18 Jun 18 UTC

closed 01:49PM - 18 Jun 18 UTC

jcmcnch

Hi, I've encountered a weird issue that I can't figure out - hopefully one of… you can! Here's what I did: 1. Imported some paired-end fastq data into qiime 2 2. Visualized the quality 3. Tried to use cutadapt: qiime cutadapt trim-paired \ --i-demultiplexed-sequences POTATOE-BACT.qza \ --p-front-f CCTACGGGNGGCWGCAG \ --p-front-r GACTACHVGGGTATCTAATCC \ --o-trimmed-sequences POTATOE-BACT_primers_trimmed.qza \ --verbose I noticed the issue when I looked at the standard output from this command. There were 6 files out of 37 that said no adapters were detected (in this case primers). I checked manually, and sure enough they were there in the fastq files. A simple grep search showed that most of the reads had the fwd/rev primer in them. So, then I tried importing two of the samples separately (1 that worked and 1 that didn't). This time, the issue didn't occur... the file that failed before had its primers detected successfully. I've also confirmed that cutadapt standalone does not have this issue. I have included some of the log information below. I'm running the same cutadapt that qiime has access to (qiime2-2018.2, installed from conda, both were run from same environment). As far as I can tell the options are more or less the same, so I'm not sure how I am getting this unpredictable behaviour. Upon closer inspection of the logs, it appears that somehow the CLI arguments are getting messed up such that R1 and R2 are switched in these samples in qiime2 (see below) which would explain why it's not finding primers. I don't understand how this can be the case as the manifest file I used for the initial batch test looks fine to me (pasted at the very bottom) and it works for the majority of samples. And I also ran another test where I pulled out one of the offending files from the qza archive and ran it with standalone cutadapt and didn't get this issue. So to me, it seems like qiime2 is importing it correctly according to the manifest but somehow the instructions to cutadapt are getting garbled. But I would love this to be a simple mistake on my part but I just can't see where it is... Thanks in advance for your help, Jesse Log for failed file (batch import of 37 samples to qiime2 then cutadapt run on all together with qiime2): ``` This is cutadapt 1.15 with Python 3.5.5 Command line parameters: --cores 1 --error-rate 0.1 --times 1 --overlap 3 -o /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-r6zhwz2c/stationP5_41_L001_R2_001.fastq.gz -p /tmp/q2-CasavaOneEightSingleLanePerSampleDirFmt-r6zhwz2c/stationP5_4_L001_R1_001.fastq.gz --front CCTACGGGNGGCWGCAG -G GACTACHVGGGTATCTAATCC /tmp/qiime2-archive-gwcyl56w/08e4e960-f54e-4038-86e5-486610afe00b/data/stationP5_41_L001_R2_001.fastq.gz /tmp/qiime2-archive-gwcyl56w/08e4e960-f54e-4038-86e5-486610afe00b/data/stationP5_4_L001_R1_001.fastq.gz Running on 1 core Trimming 2 adapters with at most 10.0% errors in paired-end mode ... Finished in 18.84 s (111 us/read; 0.54 M reads/minute). === Summary === Total read pairs processed: 170,296 Read 1 with adapter: 3 (0.0%) Read 2 with adapter: 6 (0.0%) Pairs written (passing filters): 170,296 (100.0%) Total basepairs processed: 85,488,592 bp Read 1: 42,744,296 bp Read 2: 42,744,296 bp Total written (filtered): 85,488,496 bp (100.0%) Read 1: 42,744,254 bp Read 2: 42,744,242 bp === First read: Adapter 1 === Sequence: CCTACGGGNGGCWGCAG; Type: regular 5'; Length: 17; Trimmed: 3 times. No. of allowed errors: 0-9 bp: 0; 10-17 bp: 1 Overview of removed sequences length count expect max.err error counts 8 1 2.6 0 1 17 2 0.0 1 2 === Second read: Adapter 2 === Sequence: GACTACHVGGGTATCTAATCC; Type: regular 5'; Length: 21; Trimmed: 6 times. No. of allowed errors: 0-9 bp: 0; 10-19 bp: 1; 20-21 bp: 2 Overview of removed sequences length count expect max.err error counts 3 4 2660.9 0 4 21 2 0.0 2 0 2 ``` Log for successful run where I imported the failed file in a smaller batch (only 2 samples) and it worked inexplicably: ``` This is cutadapt 1.15 with Python 3.5.5 Command line parameters: -a CCTACGGGNGGCWGCAG -A GACTACHVGGGTATCTAATCC -o stationP5outR1.fastq.gz -p stationP5outR2.fastq.gz stationP5_4_L001_R1_001.fastq.gz stationP5_41_L001_R2_001.fastq.gz Running on 1 core Trimming 2 adapters with at most 10.0% errors in paired-end mode ... Finished in 14.72 s (86 us/read; 0.69 M reads/minute). === Summary === Total read pairs processed: 170,296 Read 1 with adapter: 169,781 (99.7%) Read 2 with adapter: 169,584 (99.6%) Pairs written (passing filters): 170,296 (100.0%) Total basepairs processed: 85,488,592 bp Read 1: 42,744,296 bp Read 2: 42,744,296 bp Total written (filtered): 312,231 bp (0.4%) Read 1: 131,759 bp Read 2: 180,472 bp === First read: Adapter 1 === Sequence: CCTACGGGNGGCWGCAG; Type: regular 3'; Length: 17; Trimmed: 169781 times. No. of allowed errors: 0-9 bp: 0; 10-17 bp: 1 Bases preceding removed adapters: A: 0.0% C: 0.0% G: 0.0% T: 0.0% none/other: 100.0% Overview of removed sequences length count expect max.err error counts 3 6 2660.9 0 6 4 4 665.2 0 4 250 18 0.0 1 15 3 251 169753 0.0 1 162055 7698 === Second read: Adapter 2 === Sequence: GACTACHVGGGTATCTAATCC; Type: regular 3'; Length: 21; Trimmed: 169584 times. No. of allowed errors: 0-9 bp: 0; 10-19 bp: 1; 20-21 bp: 2 Bases preceding removed adapters: A: 0.0% C: 0.0% G: 0.0% T: 0.0% none/other: 100.0% Overview of removed sequences length count expect max.err error counts 3 4 2660.9 0 4 5 1 166.3 0 1 9 2 0.6 0 0 2 244 1 0.0 2 1 248 4 0.0 2 0 0 4 250 19 0.0 2 10 9 251 169553 0.0 2 164116 5030 407 ``` Log for successful run of same file with standalone cutadapt: ``` This is cutadapt 1.15 with Python 3.5.5 Command line parameters: --discard-untrimmed -g CCTACGGGNGGCWGCAG -G GACTACHVGGGTATCTAATCC -o 3237-WHJ-0005_S5_L001_R1_primers-trimmed.fastq.gz -p 3237-WHJ-0005_S5_L001_R2_primers-trimmed.fastq.gz 3237-WHJ-0005_S5_L001_R1_001.fastq.gz 3237-WHJ-0005_S5_L001_R2_001.fastq.gz Running on 1 core Trimming 2 adapters with at most 10.0% errors in paired-end mode ... Finished in 14.24 s (84 us/read; 0.72 M reads/minute). === Summary === Total read pairs processed: 170,296 Read 1 with adapter: 169,895 (99.8%) Read 2 with adapter: 169,605 (99.6%) Pairs written (passing filters): 169,282 (99.4%) Total basepairs processed: 85,488,592 bp Read 1: 42,744,296 bp Read 2: 42,744,296 bp Total written (filtered): 78,550,377 bp (91.9%) Read 1: 39,613,870 bp Read 2: 38,936,507 bp === First read: Adapter 1 === Sequence: CCTACGGGNGGCWGCAG; Type: regular 5'; Length: 17; Trimmed: 169895 times. No. of allowed errors: 0-9 bp: 0; 10-17 bp: 1 Overview of removed sequences length count expect max.err error counts 3 85 2660.9 0 85 11 1 0.0 1 1 14 4 0.0 1 2 2 15 24 0.0 1 9 15 16 943 0.0 1 203 740 17 168558 0.0 1 162055 6503 18 280 0.0 1 15 265 === Second read: Adapter 2 === Sequence: GACTACHVGGGTATCTAATCC; Type: regular 5'; Length: 21; Trimmed: 169605 times. No. of allowed errors: 0-9 bp: 0; 10-19 bp: 1; 20-21 bp: 2 Overview of removed sequences length count expect max.err error counts 15 2 0.0 1 0 2 16 3 0.0 1 0 3 17 13 0.0 1 10 3 18 20 0.0 1 3 17 19 103 0.0 1 4 5 94 20 1748 0.0 2 86 1604 58 21 167289 0.0 2 164116 2972 201 22 420 0.0 2 10 379 31 23 2 0.0 2 0 0 2 25 4 0.0 2 0 0 4 28 1 0.0 2 1 ``` My manifest file: ``` sample-id,absolute-filepath,direction stationP1,$PWD/BACT-341F-805R/3237-WHJ-0001_S1_L001_R1_001.fastq.gz,forward stationP2,$PWD/BACT-341F-805R/3237-WHJ-0002_S2_L001_R1_001.fastq.gz,forward stationP3,$PWD/BACT-341F-805R/3237-WHJ-0003_S3_L001_R1_001.fastq.gz,forward stationP4,$PWD/BACT-341F-805R/3237-WHJ-0004_S4_L001_R1_001.fastq.gz,forward stationP5,$PWD/BACT-341F-805R/3237-WHJ-0005_S5_L001_R1_001.fastq.gz,forward stationP6,$PWD/BACT-341F-805R/3237-WHJ-0006_S6_L001_R1_001.fastq.gz,forward stationP7,$PWD/BACT-341F-805R/3237-WHJ-0007_S7_L001_R1_001.fastq.gz,forward stationP8,$PWD/BACT-341F-805R/3237-WHJ-0008_S8_L001_R1_001.fastq.gz,forward stationP9,$PWD/BACT-341F-805R/3237-WHJ-0009_S9_L001_R1_001.fastq.gz,forward stationP10,$PWD/BACT-341F-805R/3237-WHJ-0010_S10_L001_R1_001.fastq.gz,forward stationP11,$PWD/BACT-341F-805R/3237-WHJ-0011_S11_L001_R1_001.fastq.gz,forward stationP12,$PWD/BACT-341F-805R/3237-WHJ-0012_S12_L001_R1_001.fastq.gz,forward stationP13,$PWD/BACT-341F-805R/3237-WHJ-0013_S13_L001_R1_001.fastq.gz,forward stationP14,$PWD/BACT-341F-805R/3237-WHJ-0014_S14_L001_R1_001.fastq.gz,forward stationP15,$PWD/BACT-341F-805R/3237-WHJ-0015_S15_L001_R1_001.fastq.gz,forward stationP16,$PWD/BACT-341F-805R/3237-WHJ-0016_S16_L001_R1_001.fastq.gz,forward stationP17,$PWD/BACT-341F-805R/3237-WHJ-0017_S17_L001_R1_001.fastq.gz,forward stationP18,$PWD/BACT-341F-805R/3237-WHJ-0018_S18_L001_R1_001.fastq.gz,forward stationP20,$PWD/BACT-341F-805R/3237-WHJ-0019_S19_L001_R1_001.fastq.gz,forward stationP21,$PWD/BACT-341F-805R/3237-WHJ-0020_S20_L001_R1_001.fastq.gz,forward stationP22,$PWD/BACT-341F-805R/3237-WHJ-0021_S21_L001_R1_001.fastq.gz,forward stationP23,$PWD/BACT-341F-805R/3237-WHJ-0022_S22_L001_R1_001.fastq.gz,forward stationP24,$PWD/BACT-341F-805R/3237-WHJ-0023_S23_L001_R1_001.fastq.gz,forward stationP25,$PWD/BACT-341F-805R/3237-WHJ-0024_S24_L001_R1_001.fastq.gz,forward stationP26,$PWD/BACT-341F-805R/3237-WHJ-0025_S25_L001_R1_001.fastq.gz,forward stationP27,$PWD/BACT-341F-805R/3237-WHJ-0026_S26_L001_R1_001.fastq.gz,forward stationP28,$PWD/BACT-341F-805R/3237-WHJ-0027_S27_L001_R1_001.fastq.gz,forward stationP29,$PWD/BACT-341F-805R/3237-WHJ-0028_S28_L001_R1_001.fastq.gz,forward stationP30,$PWD/BACT-341F-805R/3237-WHJ-0029_S29_L001_R1_001.fastq.gz,forward stationP31,$PWD/BACT-341F-805R/3237-WHJ-0030_S30_L001_R1_001.fastq.gz,forward stationM1,$PWD/BACT-341F-805R/3237-WHJ-0031_S31_L001_R1_001.fastq.gz,forward stationM2,$PWD/BACT-341F-805R/3237-WHJ-0032_S32_L001_R1_001.fastq.gz,forward stationM3,$PWD/BACT-341F-805R/3237-WHJ-0033_S33_L001_R1_001.fastq.gz,forward stationM4,$PWD/BACT-341F-805R/3237-WHJ-0034_S34_L001_R1_001.fastq.gz,forward stationM5,$PWD/BACT-341F-805R/3237-WHJ-0035_S35_L001_R1_001.fastq.gz,forward stationM6,$PWD/BACT-341F-805R/3237-WHJ-0036_S36_L001_R1_001.fastq.gz,forward stationM7,$PWD/BACT-341F-805R/3237-WHJ-0037_S37_L001_R1_001.fastq.gz,forward stationP1,$PWD/BACT-341F-805R/3237-WHJ-0001_S1_L001_R2_001.fastq.gz,reverse stationP2,$PWD/BACT-341F-805R/3237-WHJ-0002_S2_L001_R2_001.fastq.gz,reverse stationP3,$PWD/BACT-341F-805R/3237-WHJ-0003_S3_L001_R2_001.fastq.gz,reverse stationP4,$PWD/BACT-341F-805R/3237-WHJ-0004_S4_L001_R2_001.fastq.gz,reverse stationP5,$PWD/BACT-341F-805R/3237-WHJ-0005_S5_L001_R2_001.fastq.gz,reverse stationP6,$PWD/BACT-341F-805R/3237-WHJ-0006_S6_L001_R2_001.fastq.gz,reverse stationP7,$PWD/BACT-341F-805R/3237-WHJ-0007_S7_L001_R2_001.fastq.gz,reverse stationP8,$PWD/BACT-341F-805R/3237-WHJ-0008_S8_L001_R2_001.fastq.gz,reverse stationP9,$PWD/BACT-341F-805R/3237-WHJ-0009_S9_L001_R2_001.fastq.gz,reverse stationP10,$PWD/BACT-341F-805R/3237-WHJ-0010_S10_L001_R2_001.fastq.gz,reverse stationP11,$PWD/BACT-341F-805R/3237-WHJ-0011_S11_L001_R2_001.fastq.gz,reverse stationP12,$PWD/BACT-341F-805R/3237-WHJ-0012_S12_L001_R2_001.fastq.gz,reverse stationP13,$PWD/BACT-341F-805R/3237-WHJ-0013_S13_L001_R2_001.fastq.gz,reverse stationP14,$PWD/BACT-341F-805R/3237-WHJ-0014_S14_L001_R2_001.fastq.gz,reverse stationP15,$PWD/BACT-341F-805R/3237-WHJ-0015_S15_L001_R2_001.fastq.gz,reverse stationP16,$PWD/BACT-341F-805R/3237-WHJ-0016_S16_L001_R2_001.fastq.gz,reverse stationP17,$PWD/BACT-341F-805R/3237-WHJ-0017_S17_L001_R2_001.fastq.gz,reverse stationP18,$PWD/BACT-341F-805R/3237-WHJ-0018_S18_L001_R2_001.fastq.gz,reverse stationP20,$PWD/BACT-341F-805R/3237-WHJ-0019_S19_L001_R2_001.fastq.gz,reverse stationP21,$PWD/BACT-341F-805R/3237-WHJ-0020_S20_L001_R2_001.fastq.gz,reverse stationP22,$PWD/BACT-341F-805R/3237-WHJ-0021_S21_L001_R2_001.fastq.gz,reverse stationP23,$PWD/BACT-341F-805R/3237-WHJ-0022_S22_L001_R2_001.fastq.gz,reverse stationP24,$PWD/BACT-341F-805R/3237-WHJ-0023_S23_L001_R2_001.fastq.gz,reverse stationP25,$PWD/BACT-341F-805R/3237-WHJ-0024_S24_L001_R2_001.fastq.gz,reverse stationP26,$PWD/BACT-341F-805R/3237-WHJ-0025_S25_L001_R2_001.fastq.gz,reverse stationP27,$PWD/BACT-341F-805R/3237-WHJ-0026_S26_L001_R2_001.fastq.gz,reverse stationP28,$PWD/BACT-341F-805R/3237-WHJ-0027_S27_L001_R2_001.fastq.gz,reverse stationP29,$PWD/BACT-341F-805R/3237-WHJ-0028_S28_L001_R2_001.fastq.gz,reverse stationP30,$PWD/BACT-341F-805R/3237-WHJ-0029_S29_L001_R2_001.fastq.gz,reverse stationP31,$PWD/BACT-341F-805R/3237-WHJ-0030_S30_L001_R2_001.fastq.gz,reverse stationM1,$PWD/BACT-341F-805R/3237-WHJ-0031_S31_L001_R2_001.fastq.gz,reverse stationM2,$PWD/BACT-341F-805R/3237-WHJ-0032_S32_L001_R2_001.fastq.gz,reverse stationM3,$PWD/BACT-341F-805R/3237-WHJ-0033_S33_L001_R2_001.fastq.gz,reverse stationM4,$PWD/BACT-341F-805R/3237-WHJ-0034_S34_L001_R2_001.fastq.gz,reverse stationM5,$PWD/BACT-341F-805R/3237-WHJ-0035_S35_L001_R2_001.fastq.gz,reverse stationM6,$PWD/BACT-341F-805R/3237-WHJ-0036_S36_L001_R2_001.fastq.gz,reverse stationM7,$PWD/BACT-341F-805R/3237-WHJ-0037_S37_L001_R2_001.fastq.gz,reverse ```

My collaborator attempted the same thing as me using qiime2 and failed, and they went back to using MacQiime "extract_barcodes.py" to pull the barcodes and create the qza object. However, I do not want to use unsupported legacy software that requires root permission to install. I want to understand what is wrong here.

The warning messages in the below output are because the dual-indexing strategy uses duplicate barcodes within the R1 and R2 reads that together are unique.

Here is the command output:

Running external command line application. This may print messages to stdout and/or stderr.
The command being run is below. This command cannot be manually re-run as it will depend on temporary files that no longer exist.

Command: cutadapt --front file:/var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/tmpaexhr_tw --error-rate 0.1 --minimum-length 1 -o /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-xybzo2qn/{name}.1.fastq.gz --untrimmed-output /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-021dco45/forward.fastq.gz --pair-adapters -G file:/var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/tmpid820lh4 -p /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-xybzo2qn/{name}.2.fastq.gz --untrimmed-paired-output /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-021dco45/reverse.fastq.gz /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/qiime2-archive-qf5mbqe9/9d626484-3967-47ed-beef-162163f4ac06/data/forward.fastq.gz /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/qiime2-archive-qf5mbqe9/9d626484-3967-47ed-beef-162163f4ac06/data/reverse.fastq.gz

This is cutadapt 2.10 with Python 3.6.11
Command line parameters: --front file:/var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/tmpaexhr_tw --error-rate 0.1 --minimum-length 1 -o /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-xybzo2qn/{name}.1.fastq.gz --untrimmed-output /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-021dco45/forward.fastq.gz --pair-adapters -G file:/var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/tmpid820lh4 -p /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-xybzo2qn/{name}.2.fastq.gz --untrimmed-paired-output /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/q2-MultiplexedPairedEndBarcodeInSequenceDirFmt-021dco45/reverse.fastq.gz /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/qiime2-archive-qf5mbqe9/9d626484-3967-47ed-beef-162163f4ac06/data/forward.fastq.gz /var/folders/d5/mlx2jkz90sg6n0rn6zrf1cm5mpkgm_/T/qiime2-archive-qf5mbqe9/9d626484-3967-47ed-beef-162163f4ac06/data/reverse.fastq.gz
WARNING: Adapter 'ACTGTCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ACTGTCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ACTGTCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ACTGTCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ACTGTCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ACTGTCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ACTGTCAC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CATGCGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GACTGTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GACTGTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GACTGTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GACTGTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CACTGTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ATTGGCCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GATCTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'TCAAGCCG' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CTGATCCG' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'AAGCCCGT' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGCCCG' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'TACCCCGT' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CGTGATGA' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ACATCGTG' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GCCTAAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'TGGTCACC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CACTGTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ATTGGCCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GATCTGCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'TCAAGCCG' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CTGATCCG' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'AAGCCCGT' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GTAGCCCG' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'TACCCCGT' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CGTGATGA' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'ACATCGTG' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'GCCTAAGC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'TGGTCACC' (regular 5') was specified multiple times! Please make sure that this is what you want.
WARNING: Adapter 'CACTGTCC' (regular 5') was specified multiple times! Please make sure that this is what you want.
Processing reads on 1 core in paired-end mode ...
[---------=8 ] 00:00:57 250,000 reads @ 228.3 µs/read; 0.26 M reads/minute
Finished in 57.08 s (228 us/read; 0.26 M reads/minute).

=== Summary ===

Total read pairs processed: 250,000
Read 1 with adapter: 6,506 (2.6%)
Read 2 with adapter: 6,506 (2.6%)
Pairs written (passing filters): 0 (0.0%)

Total basepairs processed: 150,056,622 bp
Read 1: 75,184,207 bp
Read 2: 74,872,415 bp
Total written (filtered): 149,894,378 bp (99.9%)
Read 1: 75,131,846 bp
Read 2: 74,762,532 bp

=== First read: Adapter s41.NoRN ===

Sequence: ACTGTCAC; Type: regular 5'; Length: 8; Trimmed: 1842 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
3 4 3906.2 0 4
4 1 976.6 0 1
7 3 15.3 0 3
8 1832 3.8 0 1832
9 1 3.8 0 1
240 1 3.8 0 1

=== First read: Adapter s42.NoRN ===

Sequence: ACTGTCAC; Type: regular 5'; Length: 8; Trimmed: 0 times

=== First read: Adapter s43.NoRN ===

Sequence: ACTGTCAC; Type: regular 5'; Length: 8; Trimmed: 0 times

=== First read: Adapter s44.NoRN ===

Sequence: ACTGTCAC; Type: regular 5'; Length: 8; Trimmed: 0 times

=== First read: Adapter s45.NoRN ===

Sequence: ACTGTCAC; Type: regular 5'; Length: 8; Trimmed: 0 times

=== First read: Adapter s46.NoRW ===

Sequence: ACTGTCAC; Type: regular 5'; Length: 8; Trimmed: 0 times

=== First read: Adapter s47.NoRW ===

Sequence: ACTGTCAC; Type: regular 5'; Length: 8; Trimmed: 0 times

=== First read: Adapter s48.NoRW ===

Sequence: ACTGTCAC; Type: regular 5'; Length: 8; Trimmed: 0 times

=== First read: Adapter s49.NoRW ===

Sequence: GTAGTGCC; Type: regular 5'; Length: 8; Trimmed: 1353 times

No. of allowed errors:
0-8 bp: 0

Overview of removed sequences
length count expect max.err error counts
3 19 3906.2 0 19
4 3 976.6 0 3
6 1 61.0 0 1
7 2 15.3 0 2
8 1326 3.8 0 1326
9 1 3.8 0 1
295 1 3.8 0 1

=== First read: Adapter s50.NoRW ===