qiime dada2 denoise-ccs issues: 0 filtered sequences and return code 1

Technical Support
1

Hi qiime2 forum,

I am working with a set of 400 samples/fastq files generated using pacbio. I initially ran them on a server, using R but realized there was a small subset that had caused the job to abort. I identified the 94 fastq files the were unable to be processed, and ran them in qiime2 in my miniconda environment, v. qiime2-amplicon-2023.9.

I can provide the qza file upon request since the file is too large. I have attached the qzv summary.
samples.demux.summary.qzv (320.8 KB)

I ran the dada2 denoise command:

$ qiime dada2 denoise-ccs --i-demultiplexed-seqs ./samples.qza  --o-table dada2-ccs_table.qza  --o-representative-sequences dada2-ccs_rep.qza  --o-denoising-stats dada2-ccs_stats.qza  --p-min-len 1300 --p-max-len 1600  --p-front AGAGTTTGATCMTGGCTCAG --p-adapter TACGGYTACCTTGTTAYGACTT  --p-n-threads 10  --verbose

I was unable to complete filtering and obtained this error:

Loading required package: Rcpp
DADA2: 1.26.0 / Rcpp: 1.0.11 / RcppParallel: 5.1.6
1) Removing Primers
Read in 15050, output 0 (0%) filtered sequences.
Read in 171032, output 0 (0%) filtered sequences.
Read in 67692, output 0 (0%) filtered sequences.
Read in 149280, output 0 (0%) filtered sequences.
Read in 51936, output 0 (0%) filtered sequences.
Read in 53464, output 0 (0%) filtered sequences.
Read in 46350, output 0 (0%) filtered sequences.
Read in 13184, output 0 (0%) filtered sequences.
Read in 4854, output 0 (0%) filtered sequences.
Read in 86534, output 0 (0%) filtered sequences.
Read in 16498, output 0 (0%) filtered sequences.
Read in 89532, output 0 (0%) filtered sequences.
Read in 64320, output 0 (0%) filtered sequences.
Read in 33116, output 0 (0%) filtered sequences.
Read in 51744, output 0 (0%) filtered sequences.
Read in 128408, output 0 (0%) filtered sequences.
Read in 100100, output 0 (0%) filtered sequences.
Read in 126610, output 0 (0%) filtered sequences.
Read in 34, output 0 (0%) filtered sequences.
Read in 108540, output 0 (0%) filtered sequences.
Read in 348, output 0 (0%) filtered sequences.
Read in 29862, output 0 (0%) filtered sequences.
Read in 6024, output 0 (0%) filtered sequences.
Read in 270, output 0 (0%) filtered sequences.
Read in 10156, output 0 (0%) filtered sequences.
Read in 34046, output 0 (0%) filtered sequences.
Read in 2212, output 0 (0%) filtered sequences.
Read in 215912, output 0 (0%) filtered sequences.
Read in 124, output 0 (0%) filtered sequences.
Read in 17382, output 0 (0%) filtered sequences.
Read in 38, output 0 (0%) filtered sequences.
Read in 37208, output 0 (0%) filtered sequences.
Read in 166204, output 0 (0%) filtered sequences.
Read in 3650, output 0 (0%) filtered sequences.
Read in 26806, output 0 (0%) filtered sequences.
Read in 155250, output 0 (0%) filtered sequences.
Multiple matches to the primer(s) in some sequences. Using the longest possible match.
20572 sequences out of 80682 are being reverse-complemented.
Read in 80682, output 34 (0%) filtered sequences.
778 sequences out of 2670 are being reverse-complemented.
Error in sapply(match.fwd, end) + 1 :
  non-numeric argument to binary operator
3: removePrimers(unfilts, nop, primer, dada2:::rc(primerR), max.mismatch = maxMismatch,
       allow.indels = indels, orient = TRUE, verbose = TRUE)
2: withCallingHandlers(expr, warning = function(w) if (inherits(w,
       classes)) tryInvokeRestart("muffleWarning"))
1: suppressWarnings(removePrimers(unfilts, nop, primer, dada2:::rc(primerR),
       max.mismatch = maxMismatch, allow.indels = indels, orient = TRUE,
       verbose = TRUE))
Traceback (most recent call last):
  File "/home/angelica/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 440, in denoise_ccs
    run_commands([cmd])
  File "/home/angelica/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 36, in run_commands
    subprocess.run(cmd, check=True)
  File "/home/angelica/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/subprocess.py", line 516, in run
    raise CalledProcessError(retcode, process.args,
subprocess.CalledProcessError: Command '['run_dada.R', '--input_directory', '/tmp/qiime2/angelica/data/52be9217-5855-4711-82ee-c2eadbc54f85/data', '--output_path', '/tmp/tmpcb2fx7km/output.tsv.biom', '--output_track', '/tmp/tmpcb2fx7km/track.tsv', '--removed_primer_directory', '/tmp/tmpcb2fx7km/nop', '--filtered_directory', '/tmp/tmpcb2fx7km/filt', '--forward_primer', 'AGAGTTTGATCMTGGCTCAG', '--reverse_primer', 'TACGGYTACCTTGTTAYGACTT', '--max_mismatch', '2', '--indels', 'False', '--truncation_length', '0', '--trim_left', '0', '--max_expected_errors', '2.0', '--truncation_quality_score', '2', '--min_length', '1300', '--max_length', '1600', '--pooling_method', 'independent', '--chimera_method', 'consensus', '--min_parental_fold', '3.5', '--allow_one_off', 'False', '--num_threads', '12', '--learn_min_reads', '1000000', '--homopolymer_gap_penalty', 'NULL', '--band_size', '32']' returned non-zero exit status 1.

During handling of the above exception, another exception occurred:

Traceback (most recent call last):
  File "/home/angelica/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2cli/commands.py", line 520, in __call__
    results = self._execute_action(
  File "/home/angelica/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2cli/commands.py", line 581, in _execute_action
    results = action(**arguments)
  File "<decorator-gen-79>", line 2, in denoise_ccs
  File "/home/angelica/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/qiime2/sdk/action.py", line 342, in bound_callable
    outputs = self._callable_executor_(
  File "/home/angelica/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/qiime2/sdk/action.py", line 566, in _callable_executor_
    output_views = self._callable(**view_args)
  File "/home/angelica/miniconda3/envs/qiime2-amplicon-2023.9/lib/python3.8/site-packages/q2_dada2/_denoise.py", line 449, in denoise_ccs
    raise Exception("An error was encountered while running DADA2"
Exception: An error was encountered while running DADA2 in R (return code 1), please inspect stdout and stderr to learn more.

I have two main problems:

  1. For most of the samples, there are 0 filtered sequences that remain after primer removal. Considering the number of reads, I'm suspicious about my primers but confused because the primers had worked fine for the other 300 samples via the server.

  2. Regarding error code 1, I used:
    qiime tools validate samples.qza
    as suggested in a previous thread here.
    This resulted in the output:
    Result samples.qza appears to be valid at level=max.

If you have any insight, I'd greatly appreciate it!

Best,
Angelica

2

Hello!
I have 0 experience with PacBio data, but could you check if the issue is with maximum length parameter - it is set to 1600, so it removes all the reads that are longer. For example, setting it to 0 will disable it.

4

Hi @timanix!

Thank you for your reply!

I tried setting max length to both:

--p-max-len 0

and

--p-max-len 1700

In both cases I still had the same errors as pasted above.

Based on the qzv, most of the sequences should have been retained with the original length,1600. See pasted below:

image
image678×450 12.5 KB

Thanks for the suggestion @timanix , it was work a try!

Best,
Angelica

7

Hi Angelica,
Can you say a little bit more about "the other 300 samples" that worked fine?
It is surprising that such a large number of samples would work fine, and then this subset of 100 (about 1/4th of your samples) just doesn't work at all.

What numbers of reads were making it through in the 300 samples that worked? Is there anything possibly different about those samples?

Additional basic info would also be helpful: Sampling environment. Pacbio instrument/chemistry. Primer set.

3 Likes
8

Hi @benjjneb,

Yes, for sure.

The entire dataset is derived from plant tissues and liquid samples derived from plants or treatments to plant tissues. All samples had been quantified for DNA prior to sequencing, and have a range of very low to very high levels of DNA.

This data was sequenced using the PacBio Revio system. This is the first time I am processing data obtained from this sequencing technology. In the past, I had used PacBio Sequel II with no problem. These samples also had plastid and mitochondrial blockers applied during sequencing.

Of the remaining ~100 samples I have yet to process, there is a mixture of low and high DNA samples. So it is possible that some of these samples will have few reads that could potentially be removed. These samples have been sequenced before with Sequel II, of which most were processed. However, they were sequenced without any blockers, so it's difficult to know whether the blockers have inferred.

I am pasting the denoising output of the 300 samples that passed this step using the server. Although there are around/or 0% filtered sequences, there are still sequences that are being reverse complemented. Is it correct to assume that is okay with pacbio ccs data? :

Multiple matches to the primer(s) in some sequences. Using the longest possible match.
41316 sequences out of 141014 are being reverse-complemented.
Read in 141014, output 18 (0%) filtered sequences.
40254 sequences out of 137770 are being reverse-complemented.
Read in 137770, output 16 (0%) filtered sequences.
39400 sequences out of 126432 are being reverse-complemented.
Read in 126432, output 18 (0%) filtered sequences.
41888 sequences out of 144842 are being reverse-complemented.
Read in 144842, output 16 (0%) filtered sequences.
43936 sequences out of 144656 are being reverse-complemented.
Read in 144656, output 22 (0%) filtered sequences.
38926 sequences out of 139628 are being reverse-complemented.
Read in 139628, output 20 (0%) filtered sequences.
42270 sequences out of 136754 are being reverse-complemented.
Read in 136754, output 10 (0%) filtered sequences.
52500 sequences out of 175006 are being reverse-complemented.
Read in 175006, output 18 (0%) filtered sequences.
41758 sequences out of 133778 are being reverse-complemented.
Read in 133778, output 18 (0%) filtered sequences.
44100 sequences out of 154732 are being reverse-complemented.
Read in 154732, output 22 (0%) filtered sequences.
44274 sequences out of 142796 are being reverse-complemented.
Read in 142796, output 22 (0%) filtered sequences.
41106 sequences out of 136814 are being reverse-complemented.
Read in 136814, output 18 (0%) filtered sequences.
36840 sequences out of 118082 are being reverse-complemented.
Read in 118082, output 24 (0%) filtered sequences.
43274 sequences out of 156554 are being reverse-complemented.
Read in 156554, output 12 (0%) filtered sequences.
38678 sequences out of 124394 are being reverse-complemented.
Read in 124394, output 20 (0%) filtered sequences.
38246 sequences out of 132248 are being reverse-complemented.
Read in 132248, output 16 (0%) filtered sequences.
43132 sequences out of 139232 are being reverse-complemented.
Read in 139232, output 20 (0%) filtered sequences.
38080 sequences out of 132282 are being reverse-complemented.
Read in 132282, output 10 (0%) filtered sequences.
41218 sequences out of 136362 are being reverse-complemented.
Read in 136362, output 22 (0%) filtered sequences.
43650 sequences out of 151292 are being reverse-complemented.
Read in 151292, output 8 (0%) filtered sequences.
45724 sequences out of 158246 are being reverse-complemented.
Read in 158246, output 24 (0%) filtered sequences.
36996 sequences out of 134272 are being reverse-complemented.
Read in 134272, output 36 (0%) filtered sequences.
54764 sequences out of 205448 are being reverse-complemented.
Read in 205448, output 8 (0%) filtered sequences.
43490 sequences out of 159552 are being reverse-complemented.
Read in 159552, output 14 (0%) filtered sequences.
47088 sequences out of 174086 are being reverse-complemented.
Read in 174086, output 20 (0%) filtered sequences.
51280 sequences out of 194340 are being reverse-complemented.
Read in 194340, output 10 (0%) filtered sequences.
49932 sequences out of 185632 are being reverse-complemented.
Read in 185632, output 18 (0%) filtered sequences.
33600 sequences out of 122922 are being reverse-complemented.
Read in 122922, output 22 (0%) filtered sequences.
33272 sequences out of 121776 are being reverse-complemented.
Read in 121776, output 32 (0%) filtered sequences.
63210 sequences out of 221042 are being reverse-complemented.
Read in 221042, output 12 (0%) filtered sequences.
40714 sequences out of 152710 are being reverse-complemented.
Read in 152710, output 24 (0%) filtered sequences.
56146 sequences out of 197800 are being reverse-complemented.
Read in 197800, output 22 (0%) filtered sequences.
41512 sequences out of 154402 are being reverse-complemented.
Read in 154402, output 24 (0%) filtered sequences.
37674 sequences out of 132176 are being reverse-complemented.
Read in 132176, output 50 (0%) filtered sequences.
47968 sequences out of 178056 are being reverse-complemented.
Read in 178056, output 14 (0%) filtered sequences.
19084 sequences out of 69282 are being reverse-complemented.
Read in 69282, output 50 (0.1%) filtered sequences.
20756 sequences out of 76298 are being reverse-complemented.
Read in 76298, output 40 (0.1%) filtered sequences.
22648 sequences out of 80272 are being reverse-complemented.
Read in 80272, output 44 (0.1%) filtered sequences.
18924 sequences out of 67498 are being reverse-complemented.
Read in 67498, output 52 (0.1%) filtered sequences.
20570 sequences out of 76156 are being reverse-complemented.
Read in 76156, output 28 (0%) filtered sequences.
19112 sequences out of 69464 are being reverse-complemented.
Read in 69464, output 50 (0.1%) filtered sequences.
23500 sequences out of 89766 are being reverse-complemented.
Read in 89766, output 44 (0%) filtered sequences.
18952 sequences out of 69600 are being reverse-complemented.
Read in 69600, output 18 (0%) filtered sequences.
20124 sequences out of 73724 are being reverse-complemented.
Read in 73724, output 60 (0.1%) filtered sequences.
25708 sequences out of 91114 are being reverse-complemented.
Read in 91114, output 32 (0%) filtered sequences.
20784 sequences out of 78646 are being reverse-complemented.
Read in 78646, output 42 (0.1%) filtered sequences.
30018 sequences out of 105054 are being reverse-complemented.
Read in 105054, output 38 (0%) filtered sequences.
23854 sequences out of 88652 are being reverse-complemented.
Read in 88652, output 24 (0%) filtered sequences.
21048 sequences out of 76754 are being reverse-complemented.
Read in 76754, output 30 (0%) filtered sequences.
22730 sequences out of 85290 are being reverse-complemented.
Read in 85290, output 20 (0%) filtered sequences.
20330 sequences out of 73752 are being reverse-complemented.
Read in 73752, output 60 (0.1%) filtered sequences.
26708 sequences out of 100804 are being reverse-complemented.
Read in 100804, output 32 (0%) filtered sequences.
18396 sequences out of 68920 are being reverse-complemented.
Read in 68920, output 36 (0.1%) filtered sequences.
28428 sequences out of 96642 are being reverse-complemented.
Read in 96642, output 34 (0%) filtered sequences.
23524 sequences out of 85700 are being reverse-complemented.
Read in 85700, output 40 (0%) filtered sequences.
35610 sequences out of 122736 are being reverse-complemented.
Read in 122736, output 20 (0%) filtered sequences.
29480 sequences out of 105706 are being reverse-complemented.
Read in 105706, output 26 (0%) filtered sequences.
35432 sequences out of 132584 are being reverse-complemented.
Read in 132584, output 20 (0%) filtered sequences.
44094 sequences out of 161574 are being reverse-complemented.
Read in 161574, output 30 (0%) filtered sequences.
36592 sequences out of 139348 are being reverse-complemented.
Read in 139348, output 20 (0%) filtered sequences.
31450 sequences out of 113368 are being reverse-complemented.
Read in 113368, output 18 (0%) filtered sequences.
35230 sequences out of 130172 are being reverse-complemented.
Read in 130172, output 26 (0%) filtered sequences.
22480 sequences out of 77536 are being reverse-complemented.
Read in 77536, output 24 (0%) filtered sequences.
32598 sequences out of 127926 are being reverse-complemented.
Read in 127926, output 36 (0%) filtered sequences.
46926 sequences out of 159636 are being reverse-complemented.
Read in 159636, output 26 (0%) filtered sequences.
36890 sequences out of 133642 are being reverse-complemented.
Read in 133642, output 26 (0%) filtered sequences.
47570 sequences out of 159758 are being reverse-complemented.
Read in 159758, output 44 (0%) filtered sequences.
44706 sequences out of 163228 are being reverse-complemented.
Read in 163228, output 6 (0%) filtered sequences.
43978 sequences out of 154916 are being reverse-complemented.
Read in 154916, output 26 (0%) filtered sequences.
49250 sequences out of 174422 are being reverse-complemented.
Read in 174422, output 24 (0%) filtered sequences.
27202 sequences out of 95798 are being reverse-complemented.
Read in 95798, output 32 (0%) filtered sequences.
41794 sequences out of 153520 are being reverse-complemented.
Read in 153520, output 26 (0%) filtered sequences.
34964 sequences out of 125064 are being reverse-complemented.
Read in 125064, output 66 (0.1%) filtered sequences.
35556 sequences out of 126050 are being reverse-complemented.
Read in 126050, output 20 (0%) filtered sequences.
36556 sequences out of 128384 are being reverse-complemented.
Read in 128384, output 36 (0%) filtered sequences.
61834 sequences out of 168316 are being reverse-complemented.
Read in 168316, output 2 (0%) filtered sequences.
Read in 15050, output 0 (0%) filtered sequences.
Read in 171032, output 0 (0%) filtered sequences.
45244 sequences out of 143534 are being reverse-complemented.
Read in 143534, output 8 (0%) filtered sequences.
11242 sequences out of 38884 are being reverse-complemented.
Read in 38884, output 10 (0%) filtered sequences.
46734 sequences out of 140216 are being reverse-complemented.
Read in 140216, output 6 (0%) filtered sequences.
Read in 67692, output 0 (0%) filtered sequences.
Read in 149280, output 0 (0%) filtered sequences.
52160 sequences out of 184740 are being reverse-complemented.
Read in 184740, output 8 (0%) filtered sequences.
55400 sequences out of 167336 are being reverse-complemented.
Read in 167336, output 8 (0%) filtered sequences.
64386 sequences out of 212124 are being reverse-complemented.
Read in 212124, output 6 (0%) filtered sequences.
48282 sequences out of 156072 are being reverse-complemented.
Read in 156072, output 4 (0%) filtered sequences.
8320 sequences out of 28486 are being reverse-complemented.
Read in 28486, output 10 (0%) filtered sequences.
Read in 51936, output 0 (0%) filtered sequences.
68868 sequences out of 244184 are being reverse-complemented.
Read in 244184, output 18 (0%) filtered sequences.
40504 sequences out of 114066 are being reverse-complemented.
Read in 114066, output 8 (0%) filtered sequences.
55642 sequences out of 200688 are being reverse-complemented.
Read in 200688, output 8 (0%) filtered sequences.
Read in 53464, output 0 (0%) filtered sequences.
15098 sequences out of 51840 are being reverse-complemented.
Read in 51840, output 8 (0%) filtered sequences.
Read in 46350, output 0 (0%) filtered sequences.
36946 sequences out of 106904 are being reverse-complemented.
Read in 106904, output 4 (0%) filtered sequences.
36274 sequences out of 113860 are being reverse-complemented.
Read in 113860, output 2 (0%) filtered sequences.
52216 sequences out of 183554 are being reverse-complemented.
Read in 183554, output 12 (0%) filtered sequences.
55202 sequences out of 198438 are being reverse-complemented.
Read in 198438, output 6 (0%) filtered sequences.
47682 sequences out of 173172 are being reverse-complemented.
Read in 173172, output 4 (0%) filtered sequences.
62122 sequences out of 211670 are being reverse-complemented.
Read in 211670, output 10 (0%) filtered sequences.
19364 sequences out of 65882 are being reverse-complemented.
Read in 65882, output 10 (0%) filtered sequences.
Read in 13184, output 0 (0%) filtered sequences.
Read in 4854, output 0 (0%) filtered sequences.
5370 sequences out of 16766 are being reverse-complemented.
Read in 16766, output 8 (0%) filtered sequences.
43688 sequences out of 141336 are being reverse-complemented.
Read in 141336, output 12 (0%) filtered sequences.
37636 sequences out of 121620 are being reverse-complemented.
Read in 121620, output 20 (0%) filtered sequences.
45198 sequences out of 172194 are being reverse-complemented.
Read in 172194, output 18 (0%) filtered sequences.
42966 sequences out of 145106 are being reverse-complemented.
Read in 145106, output 6 (0%) filtered sequences.
Read in 86534, output 0 (0%) filtered sequences.
42234 sequences out of 117156 are being reverse-complemented.
Read in 117156, output 2 (0%) filtered sequences.
40570 sequences out of 155164 are being reverse-complemented.
Read in 155164, output 36 (0%) filtered sequences.
16110 sequences out of 57828 are being reverse-complemented.
Read in 57828, output 10 (0%) filtered sequences.
49182 sequences out of 157084 are being reverse-complemented.
Read in 157084, output 26 (0%) filtered sequences.
15936 sequences out of 57758 are being reverse-complemented.
Read in 57758, output 8 (0%) filtered sequences.
40972 sequences out of 138786 are being reverse-complemented.
Read in 138786, output 24 (0%) filtered sequences.
1680 sequences out of 5760 are being reverse-complemented.
Read in 5760, output 2 (0%) filtered sequences.
Read in 16498, output 0 (0%) filtered sequences.
Read in 89532, output 0 (0%) filtered sequences.
15884 sequences out of 54098 are being reverse-complemented.
Read in 54098, output 8 (0%) filtered sequences.
12622 sequences out of 41138 are being reverse-complemented.
Read in 41138, output 10 (0%) filtered sequences.
48068 sequences out of 158050 are being reverse-complemented.
Read in 158050, output 4 (0%) filtered sequences.
48218 sequences out of 187356 are being reverse-complemented.
Read in 187356, output 8 (0%) filtered sequences.
Read in 64320, output 0 (0%) filtered sequences.
27498 sequences out of 96476 are being reverse-complemented.
Read in 96476, output 10 (0%) filtered sequences.
Read in 33116, output 0 (0%) filtered sequences.
15532 sequences out of 54526 are being reverse-complemented.
Read in 54526, output 8 (0%) filtered sequences.
Read in 51744, output 0 (0%) filtered sequences.
21294 sequences out of 73476 are being reverse-complemented.
Read in 73476, output 12 (0%) filtered sequences.
Read in 128408, output 0 (0%) filtered sequences.
52448 sequences out of 159994 are being reverse-complemented.
Read in 159994, output 4 (0%) filtered sequences.
63078 sequences out of 217868 are being reverse-complemented.
Read in 217868, output 16 (0%) filtered sequences.
22898 sequences out of 64248 are being reverse-complemented.
Read in 64248, output 2 (0%) filtered sequences.
45704 sequences out of 173126 are being reverse-complemented.
Read in 173126, output 6 (0%) filtered sequences.
18506 sequences out of 63686 are being reverse-complemented.
Read in 63686, output 14 (0%) filtered sequences.
14436 sequences out of 48808 are being reverse-complemented.
Read in 48808, output 18 (0%) filtered sequences.
38064 sequences out of 128362 are being reverse-complemented.
Read in 128362, output 8 (0%) filtered sequences.
47396 sequences out of 160180 are being reverse-complemented.
Read in 160180, output 8 (0%) filtered sequences.
56314 sequences out of 192392 are being reverse-complemented.
Read in 192392, output 4 (0%) filtered sequences.
14678 sequences out of 50370 are being reverse-complemented.
Read in 50370, output 8 (0%) filtered sequences.
62466 sequences out of 218956 are being reverse-complemented.
Read in 218956, output 8 (0%) filtered sequences.
51930 sequences out of 147612 are being reverse-complemented.
Read in 147612, output 8 (0%) filtered sequences.
52266 sequences out of 179440 are being reverse-complemented.
Read in 179440, output 6 (0%) filtered sequences.
2336 sequences out of 6240 are being reverse-complemented.
Read in 6240, output 0 (0%) filtered sequences.
64648 sequences out of 216886 are being reverse-complemented.
Read in 216886, output 6 (0%) filtered sequences.
49660 sequences out of 148064 are being reverse-complemented.
Read in 148064, output 4 (0%) filtered sequences.
55032 sequences out of 186778 are being reverse-complemented.
Read in 186778, output 6 (0%) filtered sequences.
45384 sequences out of 156160 are being reverse-complemented.
Read in 156160, output 4 (0%) filtered sequences.
67204 sequences out of 227332 are being reverse-complemented.
Read in 227332, output 14 (0%) filtered sequences.
Read in 100100, output 0 (0%) filtered sequences.
61250 sequences out of 209008 are being reverse-complemented.
Read in 209008, output 12 (0%) filtered sequences.
7802 sequences out of 24832 are being reverse-complemented.
Read in 24832, output 6 (0%) filtered sequences.
13480 sequences out of 48346 are being reverse-complemented.
Read in 48346, output 30 (0.1%) filtered sequences.
Read in 126610, output 0 (0%) filtered sequences.
5868 sequences out of 21042 are being reverse-complemented.
Read in 21042, output 8 (0%) filtered sequences.
69068 sequences out of 207058 are being reverse-complemented.
Read in 207058, output 4 (0%) filtered sequences.
35862 sequences out of 131296 are being reverse-complemented.
Read in 131296, output 36 (0%) filtered sequences.
76846 sequences out of 234550 are being reverse-complemented.
Read in 234550, output 2 (0%) filtered sequences.
Read in 34, output 0 (0%) filtered sequences.
70646 sequences out of 196186 are being reverse-complemented.
Read in 196186, output 8 (0%) filtered sequences.
15302 sequences out of 50284 are being reverse-complemented.
Read in 50284, output 12 (0%) filtered sequences.
Read in 108540, output 0 (0%) filtered sequences.
14760 sequences out of 49066 are being reverse-complemented.
Read in 49066, output 20 (0%) filtered sequences.
8700 sequences out of 27262 are being reverse-complemented.
Read in 27262, output 4 (0%) filtered sequences.
54608 sequences out of 196200 are being reverse-complemented.
Read in 196200, output 18 (0%) filtered sequences.
69306 sequences out of 207262 are being reverse-complemented.
Read in 207262, output 6 (0%) filtered sequences.
22426 sequences out of 75746 are being reverse-complemented.
Read in 75746, output 30 (0%) filtered sequences.
40518 sequences out of 146960 are being reverse-complemented.
Read in 146960, output 30 (0%) filtered sequences.
12010 sequences out of 37580 are being reverse-complemented.
Read in 37580, output 14 (0%) filtered sequences.
70952 sequences out of 211356 are being reverse-complemented.
Read in 211356, output 12 (0%) filtered sequences.
5610 sequences out of 19394 are being reverse-complemented.
Read in 19394, output 2 (0%) filtered sequences.
51264 sequences out of 175600 are being reverse-complemented.
Read in 175600, output 12 (0%) filtered sequences.
6912 sequences out of 23632 are being reverse-complemented.
Read in 23632, output 4 (0%) filtered sequences.
Read in 348, output 0 (0%) filtered sequences.
50196 sequences out of 166818 are being reverse-complemented.
Read in 166818, output 28 (0%) filtered sequences.
46244 sequences out of 166998 are being reverse-complemented.
Read in 166998, output 20 (0%) filtered sequences.
Read in 29862, output 0 (0%) filtered sequences.
Read in 6024, output 0 (0%) filtered sequences.
14470 sequences out of 49488 are being reverse-complemented.
Read in 49488, output 12 (0%) filtered sequences.
60984 sequences out of 223918 are being reverse-complemented.
Read in 223918, output 28 (0%) filtered sequences.
13868 sequences out of 45140 are being reverse-complemented.
Read in 45140, output 16 (0%) filtered sequences.
14926 sequences out of 52914 are being reverse-complemented.
Read in 52914, output 24 (0%) filtered sequences.
52286 sequences out of 197320 are being reverse-complemented.
Read in 197320, output 30 (0%) filtered sequences.
10670 sequences out of 34272 are being reverse-complemented.
Read in 34272, output 10 (0%) filtered sequences.
66502 sequences out of 188774 are being reverse-complemented.
Read in 188774, output 8 (0%) filtered sequences.
Read in 270, output 0 (0%) filtered sequences.
Read in 10156, output 0 (0%) filtered sequences.
6438 sequences out of 21548 are being reverse-complemented.
Read in 21548, output 4 (0%) filtered sequences.
18442 sequences out of 59916 are being reverse-complemented.
Read in 59916, output 24 (0%) filtered sequences.
Read in 34046, output 0 (0%) filtered sequences.
56528 sequences out of 194990 are being reverse-complemented.
Read in 194990, output 16 (0%) filtered sequences.
31598 sequences out of 95070 are being reverse-complemented.
Read in 95070, output 4 (0%) filtered sequences.
Read in 2212, output 0 (0%) filtered sequences.
48390 sequences out of 171808 are being reverse-complemented.
Read in 171808, output 20 (0%) filtered sequences.
15870 sequences out of 53588 are being reverse-complemented.
Read in 53588, output 16 (0%) filtered sequences.
Read in 215912, output 0 (0%) filtered sequences.
12674 sequences out of 40842 are being reverse-complemented.
Read in 40842, output 10 (0%) filtered sequences.
64572 sequences out of 207400 are being reverse-complemented.
Read in 207400, output 18 (0%) filtered sequences.
39794 sequences out of 148262 are being reverse-complemented.
Read in 148262, output 24 (0%) filtered sequences.
53076 sequences out of 181776 are being reverse-complemented.
Read in 181776, output 22 (0%) filtered sequences.
8540 sequences out of 28704 are being reverse-complemented.
Read in 28704, output 16 (0.1%) filtered sequences.
51586 sequences out of 177832 are being reverse-complemented.
Read in 177832, output 14 (0%) filtered sequences.
Read in 124, output 0 (0%) filtered sequences.
11656 sequences out of 39146 are being reverse-complemented.
Read in 39146, output 12 (0%) filtered sequences.
64722 sequences out of 197852 are being reverse-complemented.
Read in 197852, output 4 (0%) filtered sequences.
39664 sequences out of 145420 are being reverse-complemented.
Read in 145420, output 36 (0%) filtered sequences.
47852 sequences out of 156650 are being reverse-complemented.
Read in 156650, output 8 (0%) filtered sequences.
29892 sequences out of 105662 are being reverse-complemented.
Read in 105662, output 20 (0%) filtered sequences.
Read in 17382, output 0 (0%) filtered sequences.
Read in 38, output 0 (0%) filtered sequences.
30896 sequences out of 102118 are being reverse-complemented.
Read in 102118, output 48 (0%) filtered sequences.
Read in 37208, output 0 (0%) filtered sequences.
16462 sequences out of 55026 are being reverse-complemented.
Read in 55026, output 62 (0.1%) filtered sequences.
Read in 166204, output 0 (0%) filtered sequences.
Read in 3650, output 0 (0%) filtered sequences.
59280 sequences out of 194846 are being reverse-complemented.
Read in 194846, output 8 (0%) filtered sequences.
58756 sequences out of 185252 are being reverse-complemented.
Read in 185252, output 2 (0%) filtered sequences.
Read in 26806, output 0 (0%) filtered sequences.
57110 sequences out of 172750 are being reverse-complemented.
Read in 172750, output 12 (0%) filtered sequences.
5384 sequences out of 17136 are being reverse-complemented.
Read in 17136, output 10 (0.1%) filtered sequences.
41730 sequences out of 143426 are being reverse-complemented.
Read in 143426, output 38 (0%) filtered sequences.
6738 sequences out of 20304 are being reverse-complemented.
Read in 20304, output 4 (0%) filtered sequences.
Read in 155250, output 0 (0%) filtered sequences.
9068 sequences out of 30660 are being reverse-complemented.
Read in 30660, output 10 (0%) filtered sequences.
50278 sequences out of 158422 are being reverse-complemented.
Read in 158422, output 12 (0%) filtered sequences.
49524 sequences out of 172052 are being reverse-complemented.
Read in 172052, output 16 (0%) filtered sequences.
40616 sequences out of 140840 are being reverse-complemented.
Read in 140840, output 22 (0%) filtered sequences.
3074 sequences out of 9294 are being reverse-complemented.
Read in 9294, output 8 (0.1%) filtered sequences.
50528 sequences out of 165318 are being reverse-complemented.
Read in 165318, output 14 (0%) filtered sequences.
38520 sequences out of 135188 are being reverse-complemented.
Read in 135188, output 18 (0%) filtered sequences.
42822 sequences out of 148892 are being reverse-complemented.
Read in 148892, output 24 (0%) filtered sequences.
32508 sequences out of 112218 are being reverse-complemented.
Read in 112218, output 8 (0%) filtered sequences.
37996 sequences out of 143172 are being reverse-complemented.
Read in 143172, output 10 (0%) filtered sequences.
43314 sequences out of 158078 are being reverse-complemented.
Read in 158078, output 8 (0%) filtered sequences.
31790 sequences out of 113048 are being reverse-complemented.
Read in 113048, output 38 (0%) filtered sequences.
42966 sequences out of 157078 are being reverse-complemented.
Read in 157078, output 16 (0%) filtered sequences.
39680 sequences out of 140408 are being reverse-complemented.
Read in 140408, output 20 (0%) filtered sequences.
38496 sequences out of 131840 are being reverse-complemented.
Read in 131840, output 20 (0%) filtered sequences.
40308 sequences out of 150220 are being reverse-complemented.
Read in 150220, output 14 (0%) filtered sequences.
40638 sequences out of 138368 are being reverse-complemented.
Read in 138368, output 14 (0%) filtered sequences.
35092 sequences out of 124558 are being reverse-complemented.
Read in 124558, output 16 (0%) filtered sequences.
35284 sequences out of 129720 are being reverse-complemented.
Read in 129720, output 10 (0%) filtered sequences.
37606 sequences out of 131052 are being reverse-complemented.
Read in 131052, output 16 (0%) filtered sequences.
38206 sequences out of 131444 are being reverse-complemented.
Read in 131444, output 8 (0%) filtered sequences.
41162 sequences out of 143694 are being reverse-complemented.
Read in 143694, output 12 (0%) filtered sequences.
41970 sequences out of 155760 are being reverse-complemented.
Read in 155760, output 16 (0%) filtered sequences.
37304 sequences out of 137740 are being reverse-complemented.
Read in 137740, output 18 (0%) filtered sequences.
35222 sequences out of 130176 are being reverse-complemented.
Read in 130176, output 10 (0%) filtered sequences.
28360 sequences out of 103904 are being reverse-complemented.
Read in 103904, output 16 (0%) filtered sequences.
15376 sequences out of 50180 are being reverse-complemented.
Read in 50180, output 40 (0.1%) filtered sequences.
27246 sequences out of 97566 are being reverse-complemented.
Read in 97566, output 54 (0.1%) filtered sequences.
24958 sequences out of 88426 are being reverse-complemented.
Read in 88426, output 32 (0%) filtered sequences.
27168 sequences out of 98704 are being reverse-complemented.
Read in 98704, output 46 (0%) filtered sequences.
19338 sequences out of 68488 are being reverse-complemented.
Read in 68488, output 44 (0.1%) filtered sequences.
56064 sequences out of 189434 are being reverse-complemented.
Read in 189434, output 16 (0%) filtered sequences.
30382 sequences out of 104042 are being reverse-complemented.
Read in 104042, output 50 (0%) filtered sequences.
31342 sequences out of 116824 are being reverse-complemented.
Read in 116824, output 22 (0%) filtered sequences.
38012 sequences out of 147422 are being reverse-complemented.
Read in 147422, output 50 (0%) filtered sequences.
16210 sequences out of 58552 are being reverse-complemented.
Read in 58552, output 28 (0%) filtered sequences.
83252 sequences out of 311186 are being reverse-complemented.
Read in 311186, output 68 (0%) filtered sequences.
38388 sequences out of 145576 are being reverse-complemented.
Read in 145576, output 34 (0%) filtered sequences.
32184 sequences out of 113170 are being reverse-complemented.
Read in 113170, output 30 (0%) filtered sequences.
29314 sequences out of 102520 are being reverse-complemented.
Read in 102520, output 26 (0%) filtered sequences.
20916 sequences out of 78292 are being reverse-complemented.
Read in 78292, output 30 (0%) filtered sequences.
26082 sequences out of 99272 are being reverse-complemented.
Read in 99272, output 36 (0%) filtered sequences.
20572 sequences out of 80682 are being reverse-complemented.
Read in 80682, output 34 (0%) filtered sequences.
28620 sequences out of 103050 are being reverse-complemented.
Read in 103050, output 40 (0%) filtered sequences.
27584 sequences out of 97656 are being reverse-complemented.
Read in 97656, output 22 (0%) filtered sequences.
30364 sequences out of 83034 are being reverse-complemented.
Read in 83034, output 71690 (86.3%) filtered sequences.
35982 sequences out of 101470 are being reverse-complemented.
Read in 101470, output 83220 (82%) filtered sequences.
30698 sequences out of 83586 are being reverse-complemented.
Read in 83586, output 70736 (84.6%) filtered sequences.
37220 sequences out of 104560 are being reverse-complemented.
Read in 104560, output 83638 (80%) filtered sequences.
2082 sequences out of 5688 are being reverse-complemented.
Read in 5688, output 5158 (90.7%) filtered sequences.
31106 sequences out of 90490 are being reverse-complemented.
Read in 90490, output 75976 (84%) filtered sequences.
38954 sequences out of 105868 are being reverse-complemented.
Read in 105868, output 85778 (81%) filtered sequences.
7320 sequences out of 19066 are being reverse-complemented.
Read in 19066, output 17352 (91%) filtered sequences.
1432 sequences out of 3374 are being reverse-complemented.
Read in 3374, output 3066 (90.9%) filtered sequences.
2330 sequences out of 6342 are being reverse-complemented.
Read in 6342, output 5810 (91.6%) filtered sequences.
31464 sequences out of 88086 are being reverse-complemented.
Read in 88086, output 76614 (87%) filtered sequences.
41562 sequences out of 113632 are being reverse-complemented.
Read in 113632, output 95216 (83.8%) filtered sequences.
5572 sequences out of 14616 are being reverse-complemented.
Read in 14616, output 13412 (91.8%) filtered sequences.
39608 sequences out of 107702 are being reverse-complemented.
Read in 107702, output 89826 (83.4%) filtered sequences.
33434 sequences out of 90872 are being reverse-complemented.
Read in 90872, output 78900 (86.8%) filtered sequences.
31288 sequences out of 83958 are being reverse-complemented.
Read in 83958, output 67978 (81%) filtered sequences.
37248 sequences out of 98208 are being reverse-complemented.
Read in 98208, output 87196 (88.8%) filtered sequences.
24188 sequences out of 68284 are being reverse-complemented.
Read in 68284, output 62270 (91.2%) filtered sequences.
11478 sequences out of 28562 are being reverse-complemented.
Read in 28562, output 26194 (91.7%) filtered sequences.
23136 sequences out of 64660 are being reverse-complemented.
Read in 64660, output 52830 (81.7%) filtered sequences.
36108 sequences out of 96790 are being reverse-complemented.
Read in 96790, output 83288 (86.1%) filtered sequences.
22040 sequences out of 61852 are being reverse-complemented.
Read in 61852, output 52956 (85.6%) filtered sequences.
23144 sequences out of 68904 are being reverse-complemented.
Read in 68904, output 55404 (80.4%) filtered sequences.
32658 sequences out of 88894 are being reverse-complemented.
Read in 88894, output 80732 (90.8%) filtered sequences.
25324 sequences out of 68552 are being reverse-complemented.
Read in 68552, output 59460 (86.7%) filtered sequences.
26210 sequences out of 75652 are being reverse-complemented.
Read in 75652, output 62844 (83.1%) filtered sequences.
6710 sequences out of 18672 are being reverse-complemented.
Read in 18672, output 17054 (91.3%) filtered sequences.
26664 sequences out of 76314 are being reverse-complemented.
Read in 76314, output 66982 (87.8%) filtered sequences.
31904 sequences out of 84278 are being reverse-complemented.
Read in 84278, output 69390 (82.3%) filtered sequences.
35900 sequences out of 97430 are being reverse-complemented.
Read in 97430, output 83134 (85.3%) filtered sequences.
27832 sequences out of 78202 are being reverse-complemented.
Read in 78202, output 69592 (89%) filtered sequences.
1308 sequences out of 3794 are being reverse-complemented.
Read in 3794, output 3480 (91.7%) filtered sequences.
2576 sequences out of 6044 are being reverse-complemented.
Read in 6044, output 5566 (92.1%) filtered sequences.
21470 sequences out of 62792 are being reverse-complemented.
Read in 62792, output 51634 (82.2%) filtered sequences.
24350 sequences out of 68084 are being reverse-complemented.
Read in 68084, output 56296 (82.7%) filtered sequences.
2368 sequences out of 6468 are being reverse-complemented.
Read in 6468, output 5942 (91.9%) filtered sequences.
32848 sequences out of 91484 are being reverse-complemented.
Read in 91484, output 74160 (81.1%) filtered sequences.
1024 sequences out of 2908 are being reverse-complemented.
Read in 2908, output 2644 (90.9%) filtered sequences.
22126 sequences out of 62374 are being reverse-complemented.
Read in 62374, output 54596 (87.5%) filtered sequences.
30364 sequences out of 86060 are being reverse-complemented.
Read in 86060, output 74210 (86.2%) filtered sequences.
25898 sequences out of 74072 are being reverse-complemented.
Read in 74072, output 64612 (87.2%) filtered sequences.
24802 sequences out of 67390 are being reverse-complemented.
Read in 67390, output 61446 (91.2%) filtered sequences.
3296 sequences out of 8506 are being reverse-complemented.
Read in 8506, output 7734 (90.9%) filtered sequences.
19930 sequences out of 77134 are being reverse-complemented.
Read in 77134, output 30 (0%) filtered sequences.
18652 sequences out of 66852 are being reverse-complemented.
Read in 66852, output 16 (0%) filtered sequences.
31138 sequences out of 123366 are being reverse-complemented.
Read in 123366, output 8 (0%) filtered sequences.
21016 sequences out of 77706 are being reverse-complemented.
Read in 77706, output 42 (0.1%) filtered sequences.
44618 sequences out of 171160 are being reverse-complemented.
Read in 171160, output 22 (0%) filtered sequences.
21908 sequences out of 78292 are being reverse-complemented.
Read in 78292, output 26 (0%) filtered sequences.
21182 sequences out of 78836 are being reverse-complemented.
Read in 78836, output 18 (0%) filtered sequences.
18484 sequences out of 64010 are being reverse-complemented.
Read in 64010, output 34 (0.1%) filtered sequences.
18426 sequences out of 64126 are being reverse-complemented.
Read in 64126, output 18 (0%) filtered sequences.
30672 sequences out of 119800 are being reverse-complemented.
Read in 119800, output 32 (0%) filtered sequences.
21218 sequences out of 77964 are being reverse-complemented.
Read in 77964, output 12 (0%) filtered sequences.
778 sequences out of 2670 are being reverse-complemented.

The error that appeared is pasted below:

Error in sapply(match.fwd, end) + 1 : 
  non-numeric argument to binary operator
Calls: removePrimers
Execution halted

The sequencing facility that processed my samples, Genome Quebec, uses a version of the 27F and 1492R primers, which they refer to as "b-version". The forward and reverse primers differ at a few bases compared to the universal full length 16S primers. I've pasted the b-version sequences below:

27b-5'-AGAGTTTGATCMTGGCTCAG-3'
1492b-5'-TACGGYTACCTTGTTAYGACTT-3'

If there's any other info I can provide, kindly let me know.

Best,
Angelica

9

OK, so the issue is that none of your samples are meaningfully passing the primer detection and removal stage. The samples "passing" are doing so with <0.1% of the initial reads.

I'd start by independently confirming that these primer sequences exist in your sequencing data, because the results of primer removal in denoise-ccs indicates that they don't. Could the data have been previously trimmed to remove primers? Could these primer sequences be not quite correct?

1 Like
10

Thanks @benjjneb
I will look into it.

I appreciate your help!

Best,
Angelica

12

Just wanted to update: indeed it was an issue with the primers. Turns out the sequence was different and was actually for the universal 16S FL primers.
Thanks for help qiime2 forum. Happy holidays!

3 Likes
13

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