PacBio DADA2-ccs plugin is losing lots of data

[Prajina-N]

Howdy,
I ran DADA2-ccs plugin for PacBio sequences for Bac16S but I am losing a lot of reads in filtering step. Search:

sample-id #q2:types	input numeric	primer-removed numeric	percentage of input primer-removed numeric	filtered numeric	percentage of input passed filter numeric	denoised numeric	non-chimeric numeric	percentage of input non-chimeric numeric
T1S1	29088	25021	86.02	17060	58.65	512	508	1.75
T1S1_2	63054	54970	87.18	38383	60.87	2060	2057	3.26
T1S2	50234	43689	86.97	30540	60.8	1596	1596	3.18
T1S2_2	70523	61689	87.47	41379	58.67	1861	1858	2.63
T1S3	69233	61022	88.14	41808	60.39	2184	2180	3.15

This is the code I ran:
#!/bin/bash

##NECESSARY JOB SPECIFICATIONS
#SBATCH --job-name=DADA2_ccs #Set the job name to "JobExample4"
#SBATCH --time=30:00:00 #Set the wall clock limit
#SBATCH --ntasks=8 #Request 1 task
#SBATCH --mem=95G #Request 2560MB (2.5GB) per node
#SBATCH --output=DADA2_ccs.%j #Send stdout/err to "Example4Out.[jobID]"

#First Executable Line

ml load Anaconda3
ml load QIIME2/2026.1-Amplicon

qiime dada2 denoise-ccs
--i-demultiplexed-seqs demux.qza
--p-min-len 1300
--p-max-len 1600
--p-max-ee 5
--p-front AGRGTTYGATYMTGGCTCAG
--p-adapter RGYTACCTTGTTACGACTT
--o-representative-sequences rep-seqs.qza
--o-denoising-stats denoising-stats.qza
--o-table table.qza
--p-n-threads 12

I have also tried different max-ee levels with little to no difference at all. This is my demux.qzv for bac 16S:

However, for ITS PacBio data, the denoising stat looks a lot better: Search:

sample-id #q2:types	input numeric	primer-removed numeric	percentage of input primer-removed numeric	filtered numeric	percentage of input passed filter numeric	denoised numeric	non-chimeric numeric	percentage of input non-chimeric numeric
T1S1	36669	32754	89.32	26279	71.67	20914	17727	48.34
T1S1_2	62619	55118	88.02	43673	69.74	32901	27308	43.61
T1S2	40653	36193	89.03	29730	73.13	22792	20879	51.36
T1S2_2	42683	37769	88.49	29678	69.53	21376	20240	47.42
T1S3	39285	34841	88.69	27769	70.69	21030	19973	50.84
T1S3_2	51719	45597	88.16	36461	70.5	26836	24498	47.37

I am basically having this problem with bacterial data. Help me please!

[ebolyen (Evan Bolyen)]

Hello @Prajina-N!

Would you be able to track down a log file for one of these runs? I think we should see the number of unique sequences printed to stdout which may be helpful in finding the issue.

[Mukhari_R (RonieM)]

Hi there, I also encountered a similar issue with by PacBio ccs reads. See below.

The command I used:

============

qiime dada2 denoise-ccs \

--i-demultiplexed-seqs os_imported-seqs.qza \

--p-min-len 1000 \

--p-max-len 1600 \

--p-max-ee 2 \

--p-front "AGRGTTYGATYMTGGCTCAG" \

--p-adapter "RGYTACCTTGTTACGACTT" \

--p-n-threads 8 \

--p-pooling-method pseudo \

--o-table os_feature-table.qza \

--o-representative-sequences os_rep-seqs.qza \

--o-denoising-stats os_denoising-stats.qza \

=========

I see that you even used a higher max-ee value, will try to use a higher one on mine to see if that helps. Additionally, I even used 'indepedent' pooling method but still came up short.

Perhaps a question for everyone, can one still proceed with downstream analyses despite this significant loss of reads after filtering?

[Prajina-N]

Hi ebolyen,
Thank you for replying. Please find the attached verbose log:

[Prajina-N]

Hi Mukhari_R,
I tried multiple max error, like 2, 5 but it did not make much difference in the reads that passed the final filter.
I am wondering if that might be the sequence quality itself. I am not entirely sure how that could happen.