cutadapt: error: [Errno 2] No such file or directory: 'file.fastq\xa0'

[Hannah1746]

I am running trim_galore on a transcriptome. There is the code that I run: trim_galore --paired Gyrinocheilus_trancriptome.1_1.fastq Gyrinocheilus_trancriptome.1_2.fastq

This is what is written in the terminal: Multicore support not enabled. Proceeding with single-core trimming. Path to Cutadapt set as: 'cutadapt' (default) Cutadapt seems to be working fine (tested command 'cutadapt --version') Cutadapt version: 2.3 single-core operation. No quality encoding type selected. Assuming that the data provided uses Sanger encoded Phred scores (default)

AUTO-DETECTING ADAPTER TYPE

Attempting to auto-detect adapter type from the first 1 million sequences of the first file (>> Gyrinocheilus_trancriptome.1_1.fastq <<)

Found perfect matches for the following adapter sequences: Adapter type Count Sequence Sequences analysed Percentage Illumina 187 AGATCGGAAGAGC 1000000 0.02 Nextera 9 CTGTCTCTTATA 1000000 0.00 smallRNA 2 TGGAATTCTCGG 1000000 0.00 Using Illumina adapter for trimming (count: 187). Second best hit was Nextera (count: 9)

Writing report to 'Gyrinocheilus_trancriptome.1_1.fastq_trimming_report.txt'

SUMMARISING RUN PARAMETERS

Input filename: Gyrinocheilus_trancriptome.1_1.fastq Trimming mode: paired-end Trim Galore version: 0.6.6 Cutadapt version: 2.3 Number of cores used for trimming: 1 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp

Cutadapt seems to be fairly up-to-date (version 2.3). Setting -j 1 Writing final adapter and quality trimmed output to Gyrinocheilus_trancriptome.1_1_trimmed.fq

Now performing quality (cutoff '-q 20') and adapter trimming in a single pass for the adapter sequence: 'AGATCGGAAGAGC' from file Gyrinocheilus_trancriptome.1_1.fastq <<< 10000000 sequences processed 20000000 sequences processed This is cutadapt 2.3 with Python 3.7.0 Command line parameters: -j 1 -e 0.1 -q 20 -O 1 -a AGATCGGAAGAGC Gyrinocheilus_trancriptome.1_1.fastq Processing reads on 1 core in single-end mode ... Finished in 377.63 s (16 us/read; 3.79 M reads/minute).

=== Summary ===

Total reads processed: 23,866,065 Reads with adapters: 5,264,537 (22.1%) Reads written (passing filters): 23,866,065 (100.0%)

Total basepairs processed: 2,171,811,915 bp Quality-trimmed: 5,466,756 bp (0.3%) Total written (filtered): 2,157,634,932 bp (99.3%)

=== Adapter 1 ===

Sequence: AGATCGGAAGAGC; Type: regular 3'; Length: 13; Trimmed: 5264537 times.

No. of allowed errors: 0-9 bp: 0; 10-13 bp: 1

Bases preceding removed adapters: A: 29.5% C: 26.9% G: 19.9% T: 23.7% none/other: 0.0%

Overview of removed sequences length count expect max.err error counts 1 2807456 5966516.2 0 2807456 2 2110687 1491629.1 0 2110687 3 200393 372907.3 0 200393 4 74034 93226.8 0 74034 5 51293 23306.7 0 51293 6 9812 5826.7 0 9812 7 884 1456.7 0 884 8 79 364.2 0 79 9 586 91.0 0 20 566 10 679 22.8 1 32 647 11 336 5.7 1 13 323 12 120 1.4 1 12 108 13 121 0.4 1 37 84 14 78 0.4 1 52 26 15 91 0.4 1 66 25 16 100 0.4 1 59 41 17 82 0.4 1 57 25 18 68 0.4 1 45 23 19 84 0.4 1 59 25 20 87 0.4 1 50 37 21 101 0.4 1 55 46 22 97 0.4 1 57 40 23 95 0.4 1 53 42 24 105 0.4 1 54 51 25 78 0.4 1 49 29 26 116 0.4 1 61 55 27 116 0.4 1 53 63 28 83 0.4 1 51 32 29 86 0.4 1 62 24 30 92 0.4 1 57 35 31 90 0.4 1 56 34 32 72 0.4 1 45 27 33 76 0.4 1 55 21 34 91 0.4 1 55 36 35 104 0.4 1 77 27 36 70 0.4 1 53 17 37 100 0.4 1 66 34 38 80 0.4 1 53 27 39 105 0.4 1 74 31 40 86 0.4 1 59 27 41 104 0.4 1 67 37 42 77 0.4 1 51 26 43 98 0.4 1 62 36 44 74 0.4 1 46 28 45 117 0.4 1 49 68 46 103 0.4 1 53 50 47 103 0.4 1 68 35 48 81 0.4 1 44 37 49 94 0.4 1 67 27 50 81 0.4 1 58 23 51 88 0.4 1 47 41 52 79 0.4 1 48 31 53 89 0.4 1 59 30 54 85 0.4 1 48 37 55 77 0.4 1 45 32 56 87 0.4 1 50 37 57 82 0.4 1 46 36 58 82 0.4 1 55 27 59 104 0.4 1 74 30 60 83 0.4 1 60 23 61 108 0.4 1 66 42 62 86 0.4 1 52 34 63 116 0.4 1 68 48 64 139 0.4 1 80 59 65 132 0.4 1 85 47 66 137 0.4 1 82 55 67 127 0.4 1 77 50 68 162 0.4 1 99 63 69 195 0.4 1 100 95 70 209 0.4 1 73 136 71 225 0.4 1 73 152 72 157 0.4 1 87 70 73 119 0.4 1 91 28 74 134 0.4 1 98 36 75 153 0.4 1 120 33 76 135 0.4 1 98 37 77 140 0.4 1 92 48 78 134 0.4 1 101 33 79 91 0.4 1 63 28 80 95 0.4 1 70 25 81 79 0.4 1 44 35 82 116 0.4 1 82 34 83 103 0.4 1 71 32 84 62 0.4 1 41 21 85 39 0.4 1 27 12 86 78 0.4 1 49 29 87 35 0.4 1 18 17 88 124 0.4 1 70 54 89 48 0.4 1 29 19 90 30 0.4 1 8 22 91 328 0.4 1 269 59

RUN STATISTICS FOR INPUT FILE: Gyrinocheilus_trancriptome.1_1.fastq

23866065 sequences processed in total The length threshold of paired-end sequences gets evaluated later on (in the validation step)

Writing report to 'Gyrinocheilus_trancriptome.1_2.fastq _trimming_report.txt'

SUMMARISING RUN PARAMETERS

Input filename: Gyrinocheilus_trancriptome.1_2.fastq  Trimming mode: paired-end Trim Galore version: 0.6.6 Cutadapt version: 2.3 Number of cores used for trimming: 1 Quality Phred score cutoff: 20 Quality encoding type selected: ASCII+33 Adapter sequence: 'AGATCGGAAGAGC' (Illumina TruSeq, Sanger iPCR; auto-detected) Maximum trimming error rate: 0.1 (default) Minimum required adapter overlap (stringency): 1 bp Minimum required sequence length for both reads before a sequence pair gets removed: 20 bp

Cutadapt seems to be fairly up-to-date (version 2.3). Setting -j -j 1 Writing final adapter and quality trimmed output to Gyrinocheilus_trancriptome.1_2.fastq _trimmed.fq

Now performing quality (cutoff '-q 20') and adapter trimming in a single pass for the adapter sequence: 'AGATCGGAAGAGC' from file Gyrinocheilus_trancriptome.1_2.fastq  <<< This is cutadapt 2.3 with Python 3.7.0 Command line parameters: -j 1 -e 0.1 -q 20 -O 1 -a AGATCGGAAGAGC Gyrinocheilus_trancriptome.1_2.fastq  Run "cutadapt --help" to see command-line options. See https://cutadapt.readthedocs.io/ for full documentation.

cutadapt: error: [Errno 2] No such file or directory: 'Gyrinocheilus_trancriptome.1_2.fastq\xa0'

Cutadapt terminated with exit signal: '512'. Terminating Trim Galore run, please check error message(s) to get an idea what went wrong...

It seems like the first of the pair-reads runs fine but I don't understand why the second is failing.

[Hannah1746]

Just re run the code and it is working fine now...

[FelixKrueger]

Good to hear. My guess would have been that you accidentally 'Gyrinocheilus_trancriptome.1_2.fastq\xa0' appended something weird to the end of the filename for R2. Let's just forget about it though :) Best of luck!