# High number of unique sequences in PacBio full-length 16S CCS data with DADA2
**URL:** https://forum.qiime2.org/t/high-number-of-unique-sequences-in-pacbio-full-length-16s-ccs-data-with-dada2/34043
**Category:** Other Bioinformatics Tools
**Tags:** dada2
**Created:** [March 9, 2026, 12:09pm UTC](https://forum.qiime2.org/t/high-number-of-unique-sequences-in-pacbio-full-length-16s-ccs-data-with-dada2/34043 "2026-03-09T12:09:38Z")
**Posts on this page:** 3
**Page:** 1
### Author:  [@Muhammad\_Ali](https://forum.qiime2.org/u/Muhammad_Ali)
#### Post date: [March 9, 2026, 12:09pm UTC](https://forum.qiime2.org/t/high-number-of-unique-sequences-in-pacbio-full-length-16s-ccs-data-with-dada2/34043/1 "2026-03-09T12:09:38Z")
I am analysing **PacBio Sequel II full-length 16S rRNA CCS reads (~1450 bp)** using the **DADA2 long-read workflow** and observing an unusually high number of unique sequences. During dereplication:
```auto
derepFastq()
```
almost all reads appear unique (e.g., ~11,200 unique reads from ~12,300 total reads). After denoising only a small number of reads remain.
Is such a high **unique/read ratio** normal for PacBio full-length 16S CCS data? Could this be related to **sequence orientation, primer trimming, or filtering parameters**?
Any suggestions for diagnosing or resolving this issue would be appreciated.
---
### Author:  [@colinbrislawn](https://forum.qiime2.org/u/colinbrislawn)
#### Post date: [March 12, 2026, 1:48pm UTC](https://forum.qiime2.org/t/high-number-of-unique-sequences-in-pacbio-full-length-16s-ccs-data-with-dada2/34043/2 "2026-03-12T13:48:05Z")
Hello @Muhammad_Ali,
Welcome to the forums! :qiime2:
This is a great question, as 90% of the reads being unique is unexpected for an Illumina 16S study. But is it normal for PacBio reads?
I found this repo, from when DADA2 was first being adapted for use with long, noisy data. [GitHub - benjjneb/LRASManuscript: Reproducible Analyses accompanying DADA2 + PacBio Manuscript · GitHub](https://github.com/benjjneb/LRASManuscript)
I've collected results from these 4 studies below.
[DADA2 + PacBio: ZymoBIOMICS Microbial Community Standard](https://benjjneb.github.io/LRASManuscript/LRASms_Zymo.html) - `Sample 1 - 69367 reads in 20516 unique sequences`
[DADA2 + PacBio: HMP Mock Community](https://benjjneb.github.io/LRASManuscript/LRASms_HMP.html) -
`Sample 1 - 69963 reads in 19873 unique sequences.`
[DADA2 + PacBio: Fecal Samples](https://benjjneb.github.io/LRASManuscript/LRASms_fecal.html) -
```auto
## Sample 1 - 17147 reads in 4468 unique sequences.
## Sample 2 - 26190 reads in 4958 unique sequences.
## Sample 3 - 24081 reads in 5326 unique sequences.
## Sample 4 - 16474 reads in 3668 unique sequences.
## Sample 5 - 28171 reads in 6385 unique sequences.
## Sample 6 - 24453 reads in 5529 unique sequences.
## Sample 7 - 21411 reads in 5519 unique sequences.
## Sample 8 - 18429 reads in 5152 unique sequences.
## Sample 9 - 13258 reads in 3772 unique sequences.
```
[DADA2 + PacBio: S. aureus from Wagner et al. 2016](https://benjjneb.github.io/LRASManuscript/LRASms_Saureus.html) -
`Sample 1 - 4056 reads in 1457 unique sequences.`
Yes, these number seem high. Something could be wrong.
> [@Muhammad\_Ali](#):
>
> Could this be related to **sequence orientation, primer trimming, or filtering parameters**?
Good thinking! Out of all of these, extra primers are guaranteed to make more of the reads unique. Maybe the adapters could be cut off with **cutadapt**?
> **[cutadapt trim-single - QIIME 2 Library](https://library.qiime2.org/plugins/qiime2/q2-cutadapt/overview#q2-action-cutadapt-trim-single)**
>
> This QIIME 2 plugin uses cutadapt to work with adapters (e.g. barcodes, primers) in sequence data.
Good luck! Let us know what you try next!
* * *
Looks like this was cross posted on the DADA2 GitHub: [PacBio full-length 16S CCS data: almost all reads unique during dereplication and very few reads retained after denoising · Issue #2182 · benjjneb/dada2 · GitHub](https://github.com/benjjneb/dada2/issues/2182)
---
### Author:  [@cjfields](https://forum.qiime2.org/u/cjfields)
#### Post date: [April 24, 2026, 5:20am UTC](https://forum.qiime2.org/t/high-number-of-unique-sequences-in-pacbio-full-length-16s-ccs-data-with-dada2/34043/3 "2026-04-24T05:20:06Z")
How many rounds of PCR amplification have these been through? Also, are these filtered to remove reads with less than 99.9% read accuracy? We’ve found these both tend to look like they are from extremely diverse environments where ~95% of reads are effectively unique within a sample.