# \#dada2 **URL:** https://forum.qiime2.org/tag/dada2/2.md [Latest](https://forum.qiime2.org/latest.md) · [Categories](https://forum.qiime2.org/categories.md) · [Tags](https://forum.qiime2.org/tags.md) --- ## [CASAVA format BC distance error](https://forum.qiime2.org/t/casava-format-bc-distance-error/34350)
**Author:** [@Ashoi\_Dantra](https://forum.qiime2.org/u/Ashoi_Dantra)\ **Replies:** 6\ **Last updated:** [October 12, 2026, 9:13pm UTC](https://forum.qiime2.org/t/casava-format-bc-distance-error/34350 "2026-10-12T21:13:23Z")
I am working with 16S rRNA gene amplicon sequencing data in QIIME 2 (version 2026.4), installed through Conda on an HPC cluster. I have generated beta-diversity distance matrices using Bray–Curtis dissimilarity and Jacc… --- ## [PacBio ITS workflow - some questions](https://forum.qiime2.org/t/pacbio-its-workflow-some-questions/34263)
**Author:** [@Rob\_DNA](https://forum.qiime2.org/u/Rob_DNA)\ **Replies:** 4\ **Last updated:** [September 9, 2026, 9:41am UTC](https://forum.qiime2.org/t/pacbio-its-workflow-some-questions/34263 "2026-09-09T09:41:44Z")
Hello, I've use QIIME2 quite a lot for Illumina paired end data, but now it is the first time I'll use it with PacBio full ITS data. The primers used are ITS9mun (GTACACACCGCCCGTCG) and ITS4ngsUni (CGCCTSCSCTTANTDATATGC… --- ## [Very low percentage of merged reads in DADA2 with 16S V4, 2×150 bp paired-end sequencing](https://forum.qiime2.org/t/very-low-percentage-of-merged-reads-in-dada2-with-16s-v4-2x150-bp-paired-end-sequencing/34302)
**Author:** [@Sergio](https://forum.qiime2.org/u/Sergio)\ **Replies:** 3\ **Last updated:** [August 28, 2026, 7:12pm UTC](https://forum.qiime2.org/t/very-low-percentage-of-merged-reads-in-dada2-with-16s-v4-2x150-bp-paired-end-sequencing/34302 "2026-08-28T19:12:58Z")
Question Hi, I am processing 16S rRNA amplicon sequencing data using QIIME 2 and DADA2. My samples were amplified targeting the V4 region of the 16S rRNA gene using the 515F and 806R primers. The libraries were sequenc… --- ## [Why are my reads filtered out (98%) when quality seems okay?](https://forum.qiime2.org/t/why-are-my-reads-filtered-out-98-when-quality-seems-okay/34292)
**Author:** [@lineroager](https://forum.qiime2.org/u/lineroager)\ **Replies:** 6\ **Last updated:** [August 20, 2026, 11:38am UTC](https://forum.qiime2.org/t/why-are-my-reads-filtered-out-98-when-quality-seems-okay/34292 "2026-08-20T11:38:51Z")
I am using qiime2 2026.7 on a WSL system in Ubuntu and working on paired-end 18S seq data (Illumina Miseq 2x300) from a mixed seawater community. My data is already imported and demultiplexed, but at the denoising step (… --- ## [Why truncate with dada2 when ITSxpress is an option?](https://forum.qiime2.org/t/why-truncate-with-dada2-when-itsxpress-is-an-option/34273)
**Author:** [@kranderson](https://forum.qiime2.org/u/kranderson)\ **Replies:** 16\ **Last updated:** [August 17, 2026, 3:32pm UTC](https://forum.qiime2.org/t/why-truncate-with-dada2-when-itsxpress-is-an-option/34273 "2026-08-17T15:32:18Z")
Hi! After reading through the ITSxpress tutorial, the Fungal ITS tutorial, and Rob's discussion around the current recommended fungal workflow, I've come to the conclusion that using ITSxpress is the blatantly superior o… --- ## [Request: DADA2 denoise-ccs without --p-front parameter](https://forum.qiime2.org/t/request-dada2-denoise-ccs-without-p-front-parameter/34277)
**Author:** [@Rob\_DNA](https://forum.qiime2.org/u/Rob_DNA)\ **Replies:** 2\ **Last updated:** [August 4, 2026, 7:12am UTC](https://forum.qiime2.org/t/request-dada2-denoise-ccs-without-p-front-parameter/34277 "2026-08-04T07:12:22Z")
To run dada2 denoise-ccs, you currently have to specify a --p-front which would generally be your FW primer. I assume that the main reason that this flag is required, is to re-orient the reads in the same direction, as P… --- ## [Is my truncation parameter exploration sufficient, and is my interpretation correct?](https://forum.qiime2.org/t/is-my-truncation-parameter-exploration-sufficient-and-is-my-interpretation-correct/34264)
**Author:** [@Rakaya](https://forum.qiime2.org/u/Rakaya)\ **Replies:** 1\ **Last updated:** [July 28, 2026, 3:21am UTC](https://forum.qiime2.org/t/is-my-truncation-parameter-exploration-sufficient-and-is-my-interpretation-correct/34264 "2026-07-28T03:21:23Z")
2\_trimmed.qzv (368.8 KB) Hi everyone, I’m working with 16S rRNA V3–V4 data in QIIME 2/DADA2 and tried to choose truncation parameters by systematically exploring forward and reverse truncation lengths, similar to th… --- ## [Losing reads at DADA2 Merge step 30% of my samples lost over 50% - I have tried multiple steps to fix- please help](https://forum.qiime2.org/t/losing-reads-at-dada2-merge-step-30-of-my-samples-lost-over-50-i-have-tried-multiple-steps-to-fix-please-help/34253)
**Author:** [@rdkramp](https://forum.qiime2.org/u/rdkramp)\ **Replies:** 1\ **Last updated:** [July 21, 2026, 7:50am UTC](https://forum.qiime2.org/t/losing-reads-at-dada2-merge-step-30-of-my-samples-lost-over-50-i-have-tried-multiple-steps-to-fix-please-help/34253 "2026-07-21T07:50:37Z")
Hello, I am running fish skin swabs for 16s rRNA 515F/806R sequences using Illumina MiSeq, V2, 500 cycle kit (2x250). I am having an issue with the merging step in DADA2. Around 30% of my samples have 50% loss at the m… --- ## [Can DADA2 be used with nearly uniform Q38 quality scores?](https://forum.qiime2.org/t/can-dada2-be-used-with-nearly-uniform-q38-quality-scores/34252)
**Author:** [@Katerina\_Fialova](https://forum.qiime2.org/u/Katerina_Fialova)\ **Replies:** 2\ **Last updated:** [July 21, 2026, 7:36am UTC](https://forum.qiime2.org/t/can-dada2-be-used-with-nearly-uniform-q38-quality-scores/34252 "2026-07-21T07:36:47Z")
Hi, I am analysing paired-end 16S rRNA amplicon data in QIIME 2 and would like to use DADA2 because my previous datasets were processed with the same pipeline, and I need the results to remain as comparable as possible. … --- ## [Sequencing platform fully confounded with study period: MiSeq vs NextSeq 2000 (16S rRNA V3-V4)](https://forum.qiime2.org/t/sequencing-platform-fully-confounded-with-study-period-miseq-vs-nextseq-2000-16s-rrna-v3-v4/34234)
**Author:** [@ggc](https://forum.qiime2.org/u/ggc)\ **Replies:** 3\ **Last updated:** [July 7, 2026, 2:42pm UTC](https://forum.qiime2.org/t/sequencing-platform-fully-confounded-with-study-period-miseq-vs-nextseq-2000-16s-rrna-v3-v4/34234 "2026-07-07T14:42:27Z")
Hi everyone, We're working on a nasopharyngeal 16S rRNA study (V3-V4) comparing a pre-pandemic cohort with a post-pandemic cohort in healthy individuals and individuals with invasive pneumococcal disease. Our problem is… --- ## [QIIME 2 processing of MiSeq i100 data with constant quality scores](https://forum.qiime2.org/t/qiime-2-processing-of-miseq-i100-data-with-constant-quality-scores/34199)
**Author:** [@Kat](https://forum.qiime2.org/u/Kat)\ **Replies:** 3\ **Last updated:** [June 8, 2026, 6:06pm UTC](https://forum.qiime2.org/t/qiime-2-processing-of-miseq-i100-data-with-constant-quality-scores/34199 "2026-06-08T18:06:57Z")
Hi all, Apologies for raising a question that has already been discussed in previous threads, but I am still unsure how to proceed with my dataset. Related posts: New Illumina i100 system data output question. QIIME2… --- ## [Running DADA2 on pre-processed data](https://forum.qiime2.org/t/running-dada2-on-pre-processed-data/34195)
**Author:** [@p\_auritus](https://forum.qiime2.org/u/p_auritus)\ **Replies:** 7\ **Last updated:** [June 3, 2026, 4:01am UTC](https://forum.qiime2.org/t/running-dada2-on-pre-processed-data/34195 "2026-06-03T04:01:53Z")
Hello dear QIIME 2 community :waving\_hand: I am analyzing data that was created by sequencing the 16S V3-V4 region on an Illumina NovaSeq 6000 creating 250bp paired-end reads (I am aware that sequencing this region with… --- ## [Query about false read merging or no read merging](https://forum.qiime2.org/t/query-about-false-read-merging-or-no-read-merging/34189)
**Author:** [@ahsankaleem](https://forum.qiime2.org/u/ahsankaleem)\ **Replies:** 1\ **Last updated:** [May 26, 2026, 6:59am UTC](https://forum.qiime2.org/t/query-about-false-read-merging-or-no-read-merging/34189 "2026-05-26T06:59:07Z")
Hello everyone! I hope that you are well. I have performed 2x250 bp paired-end sequencing of the V4-V5 region using a V2 500-cycle kit (7.5pM library with 20% PhiX). I got a cluster density of 1100K/mm², and a cluster p… --- ## [Merging NGS with TGS?](https://forum.qiime2.org/t/merging-ngs-with-tgs/34179)
**Author:** [@Patrick\_Wu](https://forum.qiime2.org/u/Patrick_Wu)\ **Replies:** 3\ **Last updated:** [May 20, 2026, 6:21am UTC](https://forum.qiime2.org/t/merging-ngs-with-tgs/34179 "2026-05-20T06:21:03Z")
As the title suggests, one of the clients at my core lab is asking me to analyze a batch of data generated by NGS with a new batch of data generated by TGS. Now I have done DADA2 -\> merge for two different NGS runs using… --- ## [Under the hood of the "Moving Pictures" tutorial](https://forum.qiime2.org/t/under-the-hood-of-the-moving-pictures-tutorial/34173)
**Author:** [@Phi\_Nguyen](https://forum.qiime2.org/u/Phi_Nguyen)\ **Replies:** 0\ **Last updated:** [May 13, 2026, 6:27pm UTC](https://forum.qiime2.org/t/under-the-hood-of-the-moving-pictures-tutorial/34173 "2026-05-13T18:27:41Z")
Hi QIIME 2 Community, I am a bioinformatician who has been working closely with QIIME 2. I’ve noticed that while the "Moving Pictures" tutorial is the gold standard for gaining initial experience, many newcomers struggl… --- ## [sequence alignment](https://forum.qiime2.org/t/sequence-alignment/34163)
**Author:** [@momay](https://forum.qiime2.org/u/momay)\ **Replies:** 5\ **Last updated:** [May 11, 2026, 6:54pm UTC](https://forum.qiime2.org/t/sequence-alignment/34163 "2026-05-11T18:54:33Z")
Hello, I have 16S rRNA sequencing data targeting the V3–V9 region, generated using paired-end 2×300 bp reads. I’ve been trying different truncation length combinations to improve read merging, but the merge rate consiste… --- ## [An error was encountered while running DADA2 in R (return code 1), inspect stdout and stderr to learn more](https://forum.qiime2.org/t/an-error-was-encountered-while-running-dada2-in-r-return-code-1-inspect-stdout-and-stderr-to-learn-more/34121)
**Author:** [@Zeina](https://forum.qiime2.org/u/Zeina)\ **Replies:** 10\ **Last updated:** [May 8, 2026, 10:15am UTC](https://forum.qiime2.org/t/an-error-was-encountered-while-running-dada2-in-r-return-code-1-inspect-stdout-and-stderr-to-learn-more/34121 "2026-05-08T10:15:18Z")
Hi everyone, I encountered an issue while running DADA2 pipeline on 250 environmental samples. I submitted a job to a Slurm queue on a server. qiime dada2 denoise-paired --i-demultiplexed-seqs data.qza --p-trunc-len-f … --- ## [Help - DADA2 microbiome analysis](https://forum.qiime2.org/t/help-dada2-microbiome-analysis/34161)
**Author:** [@Ana\_Lucia\_Arevalo\_Fi](https://forum.qiime2.org/u/Ana_Lucia_Arevalo_Fi)\ **Replies:** 3\ **Last updated:** [May 4, 2026, 7:11pm UTC](https://forum.qiime2.org/t/help-dada2-microbiome-analysis/34161 "2026-05-04T19:11:55Z")
Hi everyone, I’m currently working on a 16S soil microbiome dataset with ~140 samples using QIIME 2 (qiime2-amplicon-2025.7). During the DADA2 denoising step, about 25% of my samples end up with 0 reads after filtering. … --- ## [Qiime version and singletons](https://forum.qiime2.org/t/qiime-version-and-singletons/34151)
**Author:** [@Alessandro](https://forum.qiime2.org/u/Alessandro)\ **Replies:** 3\ **Last updated:** [April 27, 2026, 11:45pm UTC](https://forum.qiime2.org/t/qiime-version-and-singletons/34151 "2026-04-27T23:45:36Z")
Hello everyone, I’m running into a difference between QIIME 2 versions and I’d like to understand what’s going on. At the end of 2025, I performed 16S analyses using QIIME 2 version 2025.10.1. With that version, the fea… --- ## [High number of unique sequences in PacBio full-length 16S CCS data with DADA2](https://forum.qiime2.org/t/high-number-of-unique-sequences-in-pacbio-full-length-16s-ccs-data-with-dada2/34043)
**Author:** [@Muhammad\_Ali](https://forum.qiime2.org/u/Muhammad_Ali)\ **Replies:** 2\ **Last updated:** [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 "2026-04-24T05:20:06Z")
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: derepFastq() almost all rea… --- ## [Low overlap due to poor reverse read quality in 2×300 bp V3–V4 amplicon sequencing (DADA2)](https://forum.qiime2.org/t/low-overlap-due-to-poor-reverse-read-quality-in-2x300-bp-v3-v4-amplicon-sequencing-dada2/34122)
**Author:** [@djwbdjg](https://forum.qiime2.org/u/djwbdjg)\ **Replies:** 1\ **Last updated:** [April 13, 2026, 6:51am UTC](https://forum.qiime2.org/t/low-overlap-due-to-poor-reverse-read-quality-in-2x300-bp-v3-v4-amplicon-sequencing-dada2/34122 "2026-04-13T06:51:43Z")
Hello, I am planning to conduct 16S rRNA amplicon sequencing targeting the V3-V4 region (~460 bp) using Illumina Miseq i100 (2x300 bp paired-end) for rhizosphere samples. In theory, this setup should provide sufficient… --- ## [High but variable sequence loss (32-68%) during DADA2 filtering with V3-V4 amplicons (MiSeq 600 cycles)](https://forum.qiime2.org/t/high-but-variable-sequence-loss-32-68-during-dada2-filtering-with-v3-v4-amplicons-miseq-600-cycles/34101)
**Author:** [@Lilian\_Tiemi\_Inoue](https://forum.qiime2.org/u/Lilian_Tiemi_Inoue)\ **Replies:** 2\ **Last updated:** [April 9, 2026, 6:31am UTC](https://forum.qiime2.org/t/high-but-variable-sequence-loss-32-68-during-dada2-filtering-with-v3-v4-amplicons-miseq-600-cycles/34101 "2026-04-09T06:31:47Z")
Hi QIIME2 community, I am processing 16S rRNA data (V3-V4 region, ~464 bp amplicon) sequenced on an Illumina MiSeq using the V3 600-cycle kit (2x276 bp reads). I am seeing a significant and variable drop in the number o… --- ## [Inadequate overlap for V3-V4 (paired-end) due to quality drop](https://forum.qiime2.org/t/inadequate-overlap-for-v3-v4-paired-end-due-to-quality-drop/34006)
**Author:** [@Gorka\_Garcia](https://forum.qiime2.org/u/Gorka_Garcia)\ **Replies:** 5\ **Last updated:** [March 9, 2026, 8:14pm UTC](https://forum.qiime2.org/t/inadequate-overlap-for-v3-v4-paired-end-due-to-quality-drop/34006 "2026-03-09T20:14:38Z")
Hi everyone, I am currently analyzing 16S rRNA gene sequences (V3-V4 region) using QIIME2. I am facing a challenge during the denoising step with dada2 denoise-paired. The problem is that my sequencing quality drops si… --- ## [Integrating V3-V4 and V4 16S datasets for large-scale meta-analysis: Is Greengenes2 the optimal choice for cross-region alignment?](https://forum.qiime2.org/t/integrating-v3-v4-and-v4-16s-datasets-for-large-scale-meta-analysis-is-greengenes2-the-optimal-choice-for-cross-region-alignment/33938)
**Author:** [@wh466689](https://forum.qiime2.org/u/wh466689)\ **Replies:** 1\ **Last updated:** [March 6, 2026, 4:44pm UTC](https://forum.qiime2.org/t/integrating-v3-v4-and-v4-16s-datasets-for-large-scale-meta-analysis-is-greengenes2-the-optimal-choice-for-cross-region-alignment/33938 "2026-03-06T16:44:00Z")
Context & Motivation I am currently working on a large-scale meta-analysis involving hundreds of projects and over 10,000 samples. My dataset is a mixture of: V3-V4 region (e.g., amplicons from 341F/805R) V4 region (… --- ## [Too many elements for DADA2 - denoise in batches?](https://forum.qiime2.org/t/too-many-elements-for-dada2-denoise-in-batches/33961)
**Author:** [@niamh55](https://forum.qiime2.org/u/niamh55)\ **Replies:** 6\ **Last updated:** [March 4, 2026, 5:59pm UTC](https://forum.qiime2.org/t/too-many-elements-for-dada2-denoise-in-batches/33961 "2026-03-04T17:59:45Z")
Hi, Like some other previous posters, I’ve run into this error whilst running DADA2 “Error in table(pairdf$forward, pairdf$reverse) : attempt to make a table with \>= 2^31 elements” All primers etc have been removed an… --- ## [Analysis with DADA2... Merge Read Length](https://forum.qiime2.org/t/analysis-with-dada2-merge-read-length/34014)
**Author:** [@ebakes](https://forum.qiime2.org/u/ebakes)\ **Replies:** 1\ **Last updated:** [March 4, 2026, 4:49pm UTC](https://forum.qiime2.org/t/analysis-with-dada2-merge-read-length/34014 "2026-03-04T16:49:32Z")
Is length‑based filtering defensible? I am working with paired‑end 300 bp Illumina reads targeting the V3–V4 region. For filtering, I set truncation lengths based on the quality plots (forward reads truncated to 260 bp,… --- ## [denoise failure](https://forum.qiime2.org/t/denoise-failure/34001)
**Author:** [@Shuqi](https://forum.qiime2.org/u/Shuqi)\ **Replies:** 5\ **Last updated:** [February 24, 2026, 6:06pm UTC](https://forum.qiime2.org/t/denoise-failure/34001 "2026-02-24T18:06:22Z")
Hi QIIME2 team, I have worked with QIIME2 to analyze 16S v4-5 data for several datasets and encountered an error I have never seen before in the steps from import, demux, to denoise. Because our lab recently also had s… --- ## [Huge difference of DADA2 output with and without removing primers](https://forum.qiime2.org/t/huge-difference-of-dada2-output-with-and-without-removing-primers/33927)
**Author:** [@ivanllam](https://forum.qiime2.org/u/ivanllam)\ **Replies:** 4\ **Last updated:** [January 27, 2026, 7:24am UTC](https://forum.qiime2.org/t/huge-difference-of-dada2-output-with-and-without-removing-primers/33927 "2026-01-27T07:24:22Z")
Hi Community, I would like to gain some advices on the use of cutadapt and DADA2. I read several similar posts on the forum but my situation seems a little bit different from the previous discussion. (Qiime version: amp… --- ## [Advice in best practice for handling multiple sequencing runs and subset analyses in QIIME 2 (DADA2)](https://forum.qiime2.org/t/advice-in-best-practice-for-handling-multiple-sequencing-runs-and-subset-analyses-in-qiime-2-dada2/33913)
**Author:** [@ppm](https://forum.qiime2.org/u/ppm)\ **Replies:** 2\ **Last updated:** [January 16, 2026, 8:55am UTC](https://forum.qiime2.org/t/advice-in-best-practice-for-handling-multiple-sequencing-runs-and-subset-analyses-in-qiime-2-dada2/33913 "2026-01-16T08:55:26Z")
I would appreciate some advice on the best workflow for my analysis. I have two sets of samples generated from two different sequencing runs. Both runs are 16S rRNA gene amplicons (V3–V4 region, 2×300 bp). One run contai… --- ## [pair ended demultiplexed qza file causing strange error.](https://forum.qiime2.org/t/pair-ended-demultiplexed-qza-file-causing-strange-error/33876)
**Author:** [@Joely\_Corrales](https://forum.qiime2.org/u/Joely_Corrales)\ **Replies:** 1\ **Last updated:** [December 28, 2025, 8:31pm UTC](https://forum.qiime2.org/t/pair-ended-demultiplexed-qza-file-causing-strange-error/33876 "2025-12-28T20:31:43Z")
Hi, I have successfully demultiplexed this data however, as I started to denoise my data with dada2, I keep getting the error: A pair of paired-end files were found not to have the same number of records. I have tried r… [Next page](https://forum.qiime2.org/tag/dada2/2.md?match_all_tags=true&page=1&tags%5B%5D=dada2)