cutadapt parameters --p-error-rate and --p-overlap not working

When using qiime cutadapt trim-paired and changing the --p-error-rate and --p-overlap values it appears these are not being used and I am getting the same results as the default values each time. I don’t get any error messages and just noticed this by looking at the output. I am using qiime2-2019.7.

Many thanks

1 Like

Could you post your code and results out of cut_adapt? I believe using it before it’s quite conservative, so unless there’s large changes in error rate / overlap values there’s likely going to be similar results, but that’s just my exeprience. Ben

1 Like
>(qiime2-2019.7) MAC23597:Test_cutadapt lfslal$ qiime cutadapt trim-paired --i-demultiplexed-sequences demux-paired-end.qza --p-adapter-f 'AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG' --p-adapter-r 'CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT' --p-cores 2 --o-trimmed-sequences trimmed-seqs_5_4.qza --p-discard-untrimmed --p-overlap 5 --p-minimum-length 50 --verbose --p-error-rate 0.4

Running external command line application. This may print messages to stdout and/or stderr.

The commands to be run are below. These commands cannot be manually re-run as they will depend on temporary files that no longer exist.

Command: cutadapt --cores 2 --error-rate 0.4 --times 1 --overlap 5 --minimum-length 50 -o /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-52s_3z2m/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz -p /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-52s_3z2m/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz --adapter AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG -A CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT --discard-untrimmed /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-qdd946z8/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-qdd946z8/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz

>This is cutadapt 2.4 with Python 3.6.7

Command line parameters: --cores 2 --error-rate 0.4 --times 1 --overlap 5 --minimum-length 50 -o /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-52s_3z2m/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz -p /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-52s_3z2m/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz --adapter AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG -A CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT --discard-untrimmed /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-qdd946z8/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-qdd946z8/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz

WARNING: You specified a linked adapter as '-a ADAPTER1...ADAPTER2'. The
interpretation of what this means has changed in Cutadapt 2.0. (The 5'
adapter is now no longer anchored by default.) To get results consist
with the old behavior, you need to anchor the 5' adapter explicitly as
in '-a ^ADAPTER1...ADAPTER2'.

WARNING: You specified a linked adapter as '-a ADAPTER1...ADAPTER2'. The
interpretation of what this means has changed in Cutadapt 2.0. (The 5'
adapter is now no longer anchored by default.) To get results consist
with the old behavior, you need to anchor the 5' adapter explicitly as
in '-a ^ADAPTER1...ADAPTER2'.
Processing reads on 2 cores in paired-end mode ...
[8<----------] 00:00:00 16,824 reads @ 57.8 µs/read; 1.04 M reads/minute
Finished in 1.00 s (59 us/read; 1.01 M reads/minute).

=== Summary ===
Total read pairs processed: 16,824
Read 1 with adapter: 16,813 (99.9%)
Read 2 with adapter: 16,803 (99.9%)
Pairs that were too short: 27 (0.2%)
Pairs written (passing filters): 16,769 (99.7%)
Total basepairs processed: 10,094,400 bp
Read 1: 5,047,200 bp
Read 2: 5,047,200 bp
Total written (filtered): 7,417,885 bp (73.5%)
Read 1: 3,673,231 bp
Read 2: 3,744,654 bp

=== First read: Adapter 1 ===
Sequence: AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG; Type: linked; Length: 20+22; 5' trimmed: 16434 times; 3' trimmed: 16344 times
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20 bp: 2
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20-22 bp: 2
Overview of removed sequences at 5' end
length count expect max.err error counts
18 89 0.0 1 1 1 87
19 1268 0.0 1 6 564 698
20 12095 0.0 2 11597 488 10
21 2947 0.0 2 2 2073 872
22 35 0.0 2 0 1 34
Overview of removed sequences at 3' end
length count expect max.err error counts
3 13 262.9 0 13
11 1 0.0 1 1
13 5 0.0 1 5
16 26 0.0 1 24 2
17 30 0.0 1 26 4
18 82 0.0 1 76 6
21 1 0.0 2 1
34 5 0.0 2 5
35 82 0.0 2 72 9 1
36 1 0.0 2 1
37 2 0.0 2 1 0 1
39 1 0.0 2 0 1
40 1 0.0 2 1
44 2 0.0 2 2
49 2 0.0 2 2
50 88 0.0 2 77 10 1
51 5 0.0 2 4 0 1
52 1 0.0 2 1
53 3 0.0 2 2 0 1
54 2 0.0 2 2
55 7 0.0 2 6 1
56 14 0.0 2 11 3
57 47 0.0 2 43 3 1
58 24 0.0 2 18 2 4
59 91 0.0 2 83 6 2
60 87 0.0 2 76 8 3
61 635 0.0 2 554 53 28
62 1139 0.0 2 1039 70 30
63 4106 0.0 2 3805 227 74
64 6462 0.0 2 5958 374 130
65 2495 0.0 2 2311 128 56
66 628 0.0 2 574 37 17
67 58 0.0 2 53 5
68 15 0.0 2 14 0 1
69 44 0.0 2 37 4 3
70 28 0.0 2 26 0 2
71 1 0.0 2 1
72 1 0.0 2 1
73 12 0.0 2 11 1
74 33 0.0 2 32 1
78 1 0.0 2 0 1
79 25 0.0 2 22 2 1
81 1 0.0 2 1
84 1 0.0 2 1
85 1 0.0 2 1
87 1 0.0 2 1
113 7 0.0 2 0 7
287 26 0.0 2 0 0 26
290 1 0.0 2 0 0 1

=== Second read: Adapter 4 ===
Sequence: CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT; Type: linked; Length: 22+20; 5' trimmed: 16782 times; 3' trimmed: 15043 times
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20-22 bp: 2
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20 bp: 2
Overview of removed sequences at 5' end
length count expect max.err error counts
6 1 4.1 0 1
14 1 0.0 1 1
17 1 0.0 1 0 1
20 12 0.0 2 1 1 10
21 60 0.0 2 8 32 20
22 16693 0.0 2 16392 255 46
23 13 0.0 2 1 12
24 1 0.0 2 0 0 1

Overview of removed sequences at 3' end
length count expect max.err error counts
3 14 262.9 0 14
4 4 65.7 0 4
9 1 0.1 0 1
11 3 0.0 1 2 1
14 20 0.0 1 16 4
15 27 0.0 1 19 8
16 58 0.0 1 42 16
19 1 0.0 1 1
32 5 0.0 2 4 1
33 78 0.0 2 64 10 4
34 2 0.0 2 2
36 1 0.0 2 0 1
38 1 0.0 2 1
42 2 0.0 2 1 0 1
47 3 0.0 2 3
48 85 0.0 2 61 15 9
49 4 0.0 2 0 4
51 1 0.0 2 1
52 2 0.0 2 2
53 7 0.0 2 2 4 1
54 14 0.0 2 0 10 4
55 41 0.0 2 2 34 5
56 19 0.0 2 1 16 2
57 88 0.0 2 0 74 14
58 79 0.0 2 4 62 13
59 602 0.0 2 459 100 43
60 1089 0.0 2 409 593 87
61 4008 0.0 2 3245 369 394
62 6446 0.0 2 4293 1237 916
63 1571 0.0 2 505 506 560
64 551 0.0 2 219 232 100
65 54 0.0 2 22 26 6
66 12 0.0 2 8 2 2
67 41 0.0 2 27 8 6
68 26 0.0 2 22 4
69 2 0.0 2 1 1
70 1 0.0 2 1
71 11 0.0 2 0 10 1
72 32 0.0 2 4 26 2
76 1 0.0 2 0 1
77 25 0.0 2 19 4 2
79 1 0.0 2 1
82 1 0.0 2 1
83 1 0.0 2 1
85 1 0.0 2 0 1
111 7 0.0 2 7

Saved SampleData[PairedEndSequencesWithQuality] to: trimmed-seqs_5_4.qza

(qiime2-2019.7) MAC23597:Test_cutadapt lfslal$ qiime cutadapt trim-paired --i-demultiplexed-sequences demux-paired-end.qza --p-adapter-f 'AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG' --p-adapter-r 'CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT' --p-cores 2 --o-trimmed-sequences trimmed-seqs_10_1.qza --p-discard-untrimmed --p-overlap 10 --p-minimum-length 50 --verbose --p-error-rate 0.1

Running external command line application. This may print messages to stdout and/or stderr.

The commands to be run are below. These commands cannot be manually re-run as they will depend on temporary files that no longer exist.

Command: cutadapt --cores 2 --error-rate 0.1 --times 1 --overlap 10 --minimum-length 50 -o /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-5fgkeo3y/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz -p /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-5fgkeo3y/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz --adapter AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG -A CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT --discard-untrimmed /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-_sfo8409/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-_sfo8409/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz

This is cutadapt 2.4 with Python 3.6.7

Command line parameters: --cores 2 --error-rate 0.1 --times 1 --overlap 10 --minimum-length 50 -o /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-5fgkeo3y/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz -p /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-5fgkeo3y/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz --adapter AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG -A CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT --discard-untrimmed /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-_sfo8409/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz /var/folders/6_/_9q9_yg5gsdmj64pdzts83ms996x5/T/qiime2-archive-_sfo8409/719062ec-cd8e-4daa-ae52-ad00ae121800/dta/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz
WARNING: You specified a linked adapter as '-a ADAPTER1...ADAPTER2'. The
interpretation of what this means has changed in Cutadapt 2.0. (The 5'
adapter is now no longer anchored by default.) To get results consist
with the old behavior, you need to anchor the 5' adapter explicitly as
in '-a ^ADAPTER1...ADAPTER2'.
WARNING: You specified a linked adapter as '-a ADAPTER1...ADAPTER2'. The
interpretation of what this means has changed in Cutadapt 2.0. (The 5'
adapter is now no longer anchored by default.) To get results consist
with the old behavior, you need to anchor the 5' adapter explicitly as
in '-a ^ADAPTER1...ADAPTER2'.
Processing reads on 2 cores in paired-end mode ...
[8<----------] 00:00:01 16,824 reads @ 61.1 µs/read; 0.98 M reads/minute

Finished in 1.05 s (62 us/read; 0.96 M reads/minute).
=== Summary ===
Total read pairs processed: 16,824
Read 1 with adapter: 16,813 (99.9%)
Read 2 with adapter: 16,803 (99.9%)
Pairs that were too short: 27 (0.2%)
Pairs written (passing filters): 16,769 (99.7%)
Total basepairs processed: 10,094,400 bp
Read 1: 5,047,200 bp
Read 2: 5,047,200 bp
Total written (filtered): 7,417,885 bp (73.5%)
Read 1: 3,673,231 bp
Read 2: 3,744,654 bp

=== First read: Adapter 1 ===
Sequence: AAGCTCGTAGTTGAATTTCG...ATGATTAAAGGGATAGTTGGG; Type: linked; Length: 20+22; 5' trimmed: 16434 times; 3' trimmed: 16344 times
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20 bp: 2
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20-22 bp: 2
Overview of removed sequences at 5' end
length count expect max.err error counts
18 89 0.0 1 1 1 87
19 1268 0.0 1 6 564 698
20 12095 0.0 2 11597 488 10
21 2947 0.0 2 2 2073 872
22 35 0.0 2 0 1 34

Overview of removed sequences at 3' end
length count expect max.err error counts
3 13 262.9 0 13
11 1 0.0 1 1
13 5 0.0 1 5
16 26 0.0 1 24 2
17 30 0.0 1 26 4
18 82 0.0 1 76 6
21 1 0.0 2 1
34 5 0.0 2 5
35 82 0.0 2 72 9 1
36 1 0.0 2 1
37 2 0.0 2 1 0 1
39 1 0.0 2 0 1
40 1 0.0 2 1
44 2 0.0 2 2
49 2 0.0 2 2
50 88 0.0 2 77 10 1
51 5 0.0 2 4 0 1
52 1 0.0 2 1
53 3 0.0 2 2 0 1
54 2 0.0 2 2
55 7 0.0 2 6 1
56 14 0.0 2 11 3
57 47 0.0 2 43 3 1
58 24 0.0 2 18 2 4
59 91 0.0 2 83 6 2
60 87 0.0 2 76 8 3
61 635 0.0 2 554 53 28
62 1139 0.0 2 1039 70 30
63 4106 0.0 2 3805 227 74
64 6462 0.0 2 5958 374 130
65 2495 0.0 2 2311 128 56
66 628 0.0 2 574 37 17
67 58 0.0 2 53 5
68 15 0.0 2 14 0 1
69 44 0.0 2 37 4 3
70 28 0.0 2 26 0 2
71 1 0.0 2 1
72 1 0.0 2 1
73 12 0.0 2 11 1
74 33 0.0 2 32 1
78 1 0.0 2 0 1
79 25 0.0 2 22 2 1
81 1 0.0 2 1
84 1 0.0 2 1
85 1 0.0 2 1
87 1 0.0 2 1
113 7 0.0 2 0 7
287 26 0.0 2 0 0 26
290 1 0.0 2 0 0 1

=== Second read: Adapter 4 ===
Sequence: CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT; Type: linked; Length: 22+20; 5' trimmed: 16782 times; 3' trimmed: 15043 times
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20-22 bp: 2
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20 bp: 2
Overview of removed sequences at 5' end
length count expect max.err error counts
6 1 4.1 0 1
14 1 0.0 1 1
17 1 0.0 1 0 1
20 12 0.0 2 1 1 10
21 60 0.0 2 8 32 20
22 16693 0.0 2 16392 255 46
23 13 0.0 2 1 12
24 1 0.0 2 0 0 1

Overview of removed sequences at 3' end
length count expect max.err error counts
3 14 262.9 0 14
4 4 65.7 0 4
9 1 0.1 0 1
11 3 0.0 1 2 1
14 20 0.0 1 16 4
15 27 0.0 1 19 8
16 58 0.0 1 42 16
19 1 0.0 1 1
32 5 0.0 2 4 1
33 78 0.0 2 64 10 4
34 2 0.0 2 2
36 1 0.0 2 0 1
38 1 0.0 2 1
42 2 0.0 2 1 0 1
47 3 0.0 2 3
48 85 0.0 2 61 15 9
49 4 0.0 2 0 4
51 1 0.0 2 1
52 2 0.0 2 2
53 7 0.0 2 2 4 1
54 14 0.0 2 0 10 4
55 41 0.0 2 2 34 5
56 19 0.0 2 1 16 2
57 88 0.0 2 0 74 14
58 79 0.0 2 4 62 13
59 602 0.0 2 459 100 43
60 1089 0.0 2 409 593 87
61 4008 0.0 2 3245 369 394
62 6446 0.0 2 4293 1237 916
63 1571 0.0 2 505 506 560
64 551 0.0 2 219 232 100
65 54 0.0 2 22 26 6
66 12 0.0 2 8 2 2
67 41 0.0 2 27 8 6
68 26 0.0 2 22 4
69 2 0.0 2 1 1
70 1 0.0 2 1
71 11 0.0 2 0 10 1
72 32 0.0 2 4 26 2
76 1 0.0 2 0 1
77 25 0.0 2 19 4 2
79 1 0.0 2 1
82 1 0.0 2 1
83 1 0.0 2 1
85 1 0.0 2 0 1
111 7 0.0 2 7

Saved SampleData[PairedEndSequencesWithQuality] to: trimmed-seqs_10_1.qza

(qiime2-2019.7) MAC23597:Test_cutadapt lfslal$ qiime cutadapt trim-paired --i-demultiplexed-sequences demux-paired-end.qza --p-adapter-f 'AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG' --p-adapter-r 'CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT' --p-cores 2 --o-trimmed-sequences trimmed-seqs.qza --p-discard-untrimmed --p-minimum-length 50 --verbose 

Running external command line application. This may print messages to stdout and/or stderr.

The commands to be run are below. These commands cannot be manually re-run as they will depend on temporary files that no longer exist.

Command: cutadapt --cores 2 --error-rate 0.1 --times 1 --overlap 3 --minimum-length 50 -o /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-th4578gz/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz -p /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-th4578gz/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz --adapter AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG -A CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT --discard-untrimmed /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-ewimfqhs/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-ewimfqhs/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz

This is cutadapt 2.4 with Python 3.6.7

Command line parameters: --cores 2 --error-rate 0.1 --times 1 --overlap 3 --minimum-length 50 -o /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-th4578gz/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.gz -p /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/q2-CasavaOneEightSingleLanePerSampleDirFmt-th4578gz/P11F08_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz --adapter AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG -A CCCAACTATCCCTATTAATCAT...CGAAATTCAACTACGAGCTT --discard-untrimmed /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-ewimfqhs/719062ec-cd8e-4daa-ae52-ad00ae121800/data/P11F0_A10158-100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R1_001.fastq.g /var/folders/6_/_9q9_yg51gsdmj64pdzts83ms996x5/T/qiime2-archive-ewimfqhs/719062ec-cd8e-4aa-ae52-ad00ae121800/data/P11F08_A10158100_CPBGN_CGAGGCTG-AAGGCTAT_L001_R2_001.fastq.gz
WARNING: You specified a linked adapter as '-a ADAPTER1...ADAPTER2'. The
interpretation of what this means has changed in Cutadapt 2.0. (The 5'
adapter is now no longer anchored by default.) To get results consist
with the old behavior, you need to anchor the 5' adapter explicitly as
in '-a ^ADAPTER1...ADAPTER2'.
WARNING: You specified a linked adapter as '-a ADAPTER1...ADAPTER2'. The
interpretation of what this means has changed in Cutadapt 2.0. (The 5'
adapter is now no longer anchored by default.) To get results consist
with the old behavior, you need to anchor the 5' adapter explicitly as
in '-a ^ADAPTER1...ADAPTER2'.
Processing reads on 2 cores in paired-end mode ...
[8<----------] 00:00:01 16,824 reads @ 60.8 µs/read; 0.99 M reads/minute
Finished in 1.04 s (62 us/read; 0.97 M reads/minute).
=== Summary ===
Total read pairs processed: 16,824
Read 1 with adapter: 16,813 (99.9%)
Read 2 with adapter: 16,803 (99.9%)
Pairs that were too short: 27 (0.2%)
Pairs written (passing filters): 16,769 (99.7%)
Total basepairs processed: 10,094,400 bp
Read 1: 5,047,200 bp
Read 2: 5,047,200 bp
Total written (filtered): 7,417,885 bp (73.5%)
Read 1: 3,673,231 bp
Read 2: 3,744,654 bp

=== First read: Adapter 1 ===

Sequece: AAGCTCGTAGTTGAATTTCG...ATGATTAATAGGGATAGTTGGG; Type: linked; Length:20+22; 5' trimmed: 16434 times; 3' trimmed: 16344 times
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20 bp: 2
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20-22 bp: 2
Overview of removed sequences at 5' end
length count expect max.err error counts
18 89 0.0 1 1 1 87
19 1268 0.0 1 6 564 698
20 12095 0.0 2 11597 488 10
21 2947 0.0 2 2 2073 872
22 35 0.0 2 0 1 34
Overview of removed sequences at 3' end
length count expect max.err error counts
3 13 262.9 0 13
11 1 0.0 1 1
13 5 0.0 1 5
16 26 0.0 1 24 2
17 30 0.0 1 26 4
18 82 0.0 1 76 6
21 1 0.0 2 1
34 5 0.0 2 5
35 82 0.0 2 72 9 1
36 1 0.0 2 1
37 2 0.0 2 1 0 1
39 1 0.0 2 0 1
40 1 0.0 2 1
44 2 0.0 2 2
49 2 0.0 2 2
50 88 0.0 2 77 10 1
51 5 0.0 2 4 0 1
52 1 0.0 2 1
53 3 0.0 2 2 0 1
54 2 0.0 2 2
55 7 0.0 2 6 1
56 14 0.0 2 11 3
57 47 0.0 2 43 3 1
58 24 0.0 2 18 2 4
59 91 0.0 2 83 6 2
60 87 0.0 2 76 8 3
61 635 0.0 2 554 53 28
62 1139 0.0 2 1039 70 30
63 4106 0.0 2 3805 227 74
64 6462 0.0 2 5958 374 130
65 2495 0.0 2 2311 128 56
66 628 0.0 2 574 37 17
67 58 0.0 2 53 5
68 15 0.0 2 14 0 1
69 44 0.0 2 37 4 3
70 28 0.0 2 26 0 2
71 1 0.0 2 1
72 1 0.0 2 1
73 12 0.0 2 11 1
74 33 0.0 2 32 1
78 1 0.0 2 0 1
79 25 0.0 2 22 2 1
81 1 0.0 2 1
84 1 0.0 2 1
85 1 0.0 2 1
87 1 0.0 2 1
113 7 0.0 2 0 7
287 26 0.0 2 0 0 26
290 1 0.0 2 0 0 1

=== Second read: Adapter 4 ===
Sequence: CCCAACTATCCCTATTAATCAT...CAAATTCAACTACGAGCTT; Type: linked; Length: 22+20; 5' trimmed: 16782 times; 3' trimmed: 15043 times
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20-22 bp: 2
No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20 bp: 2
Overview of removed sequences at 5' end
length count expect max.err error counts6 1 4.1 0 1
14 1 0.0 1 1
17 1 0.0 1 0 1
20 12 0.0 2 1 1 10
21 60 0.0 2 8 32 20
22 16693 0.0 2 16392 255 46
23 13 0.0 2 1 12
24 1 0.0 2 0 0 1

Overview of removed sequences at 3' end
length count expect max.err error counts
3 14 262.9 0 14
4 4 65.7 0 4
9 1 0.1 0 1
11 3 0.0 1 2 1
14 20 0.0 1 16 4
15 27 0.0 1 19 8
16 58 0.0 1 42 16
19 1 0.0 1 1
32 5 0.0 2 4 1
33 78 0.0 2 64 10 4
34 2 0.0 2 2
36 1 0.0 2 0 1
38 1 0.0 2 1
42 2 0.0 2 1 0 1
47 3 0.0 2 3
48 85 0.0 2 61 15 9
49 4 0.0 2 0 4
51 1 0.0 2 1
52 2 0.0 2 2
53 7 0.0 2 2 4 1
54 14 0.0 2 0 10 4
55 41 0.0 2 2 34 5
56 19 0.0 2 1 16 2
57 88 0.0 2 0 74 14
58 79 0.0 2 4 62 13
59 602 0.0 2 459 100 43
60 1089 0.0 2 409 593 87
61 4008 0.0 2 3245 369 394
62 6446 0.0 2 4293 1237 916
63 1571 0.0 2 505 506 560
64 551 0.0 2 219 232 100
65 54 0.0 2 22 26 6
66 12 0.0 2 8 2 2
67 41 0.0 2 27 8 6
68 26 0.0 2 22 4
69 2 0.0 2 1 1
70 1 0.0 2 1
71 11 0.0 2 0 10 1
72 32 0.0 2 4 26 2
76 1 0.0 2 0 1
77 25 0.0 2 19 4 2
79 1 0.0 2 1
82 1 0.0 2 1
83 1 0.0 2 1
85 1 0.0 2 0 1
111 7 0.0 2 7
Saved SampleData[PairedEndSequencesWithQuality] to: trimmed-seqs.qza
1 Like

I’m going to need some time to review everything, thanks for posting this. Ben

Hi @SarahH!

Did you see this note here:

Try the test again, but this time only vary either the error rate, or the overlap (don’t change both - that experimental design will confound the results!). Make sure to anchor the linked fwd adapter.

If you run into additional issues, I suggest you get in touch with the cutadapt team — q2-cutadapt is simply a light wrapper around cutadapt, you can see the cutadapt commands that are being generated by q2-cutadapt for you:

Also, I suggest you spend some time reviewing the cutadapt user guide:

https://cutadapt.readthedocs.io/en/stable/guide.html