# Latest **URL:** https://forum.biobakery.org/latest.md [Latest](https://forum.biobakery.org/latest.md) · [Categories](https://forum.biobakery.org/categories.md) --- ## [Read depth and unscaled abundance in MaAsLin3](https://forum.biobakery.org/t/read-depth-and-unscaled-abundance-in-maaslin3/9067)
**Author:** [@sigrid.borte](https://forum.biobakery.org/u/sigrid.borte)\ **Replies:** 1\ **Last updated:** [September 22, 2026, 8:25pm UTC](https://forum.biobakery.org/t/read-depth-and-unscaled-abundance-in-maaslin3/9067 "2026-09-22T20:25:53Z")
I am analysing shotgun metagenomic data from approximately 9,300 stool samples using MaAsLin3. We have raw paired-end read pairs before host and quality filtering, as well as MGS count library size, defined as the sum of… --- ## [Unable to download Databases](https://forum.biobakery.org/t/unable-to-download-databases/8879)
**Author:** [@ThibaultWilmsen](https://forum.biobakery.org/u/ThibaultWilmsen)\ **Replies:** 11\ **Last updated:** [September 22, 2026, 4:14am UTC](https://forum.biobakery.org/t/unable-to-download-databases/8879 "2026-09-22T04:14:12Z")
I’m unable to download any of the databases using the commands provided in the tutorial. Has anyone encountered this issue or know what might be going wrong? humann\_databases --download chocophlan DEMO humann\_dbs human… --- ## [MaAsLin3 analysis for longitudinal clinical analysis](https://forum.biobakery.org/t/maaslin3-analysis-for-longitudinal-clinical-analysis/9065)
**Author:** [@bbernard1](https://forum.biobakery.org/u/bbernard1)\ **Replies:** 3\ **Last updated:** [September 22, 2026, 1:05am UTC](https://forum.biobakery.org/t/maaslin3-analysis-for-longitudinal-clinical-analysis/9065 "2026-09-22T01:05:40Z")
I was wondering if I could get some clarification for how to properly analyze longitudinal clinical data in MaAsLin3. Essentially, I have clinical data that is comparing the change in microbial taxon relative abundance o… --- ## [Maaslin\_contrast\_test() with median\_comparison\_abundance uses a z-test instead of t-test for linear mixed models](https://forum.biobakery.org/t/maaslin-contrast-test-with-median-comparison-abundance-uses-a-z-test-instead-of-t-test-for-linear-mixed-models/9057)
**Author:** [@Eli](https://forum.biobakery.org/u/Eli)\ **Replies:** 4\ **Last updated:** [September 15, 2026, 6:40pm UTC](https://forum.biobakery.org/t/maaslin-contrast-test-with-median-comparison-abundance-uses-a-z-test-instead-of-t-test-for-linear-mixed-models/9057 "2026-09-15T18:40:16Z")
Hello, I think I’ve found a subtle bug in maaslin3 \`maaslin\_contrast\_test()\`. For a bit of context, I had been running maaslin3 multiple times across many contrasts and then removing the duplicate contrasts after the fa… --- ## [Issue because of \`collapse\` version](https://forum.biobakery.org/t/issue-because-of-collapse-version/9061)
**Author:** [@hariszaf](https://forum.biobakery.org/u/hariszaf)\ **Replies:** 1\ **Last updated:** [September 15, 2026, 5:14pm UTC](https://forum.biobakery.org/t/issue-because-of-collapse-version/9061 "2026-09-15T17:14:39Z")
Hello. Many thanks for all the contribution! This is just to let the community know of an observation I made. Using MaAsLin 3 (maaslin3\_1.5.5) in R.4.4.1, I noticed a funny behavior caused by the collapse library. My… --- ## [Random versus fixed effects](https://forum.biobakery.org/t/random-versus-fixed-effects/9056)
**Author:** [@rriudavets](https://forum.biobakery.org/u/rriudavets)\ **Replies:** 1\ **Last updated:** [September 12, 2026, 2:47am UTC](https://forum.biobakery.org/t/random-versus-fixed-effects/9056 "2026-09-12T02:47:42Z")
Hello, First of all, thank you so much for developing MaAsLin and for your continuous help to the community! I am using MaAsLin3 to analyze my metagenomics dataset and I am a bit unsure on how to proceed with the specif… --- ## [Phylophlan Discard a few mags](https://forum.biobakery.org/t/phylophlan-discard-a-few-mags/9053)
**Author:** [@Skylady](https://forum.biobakery.org/u/Skylady)\ **Replies:** 1\ **Last updated:** [September 10, 2026, 7:17am UTC](https://forum.biobakery.org/t/phylophlan-discard-a-few-mags/9053 "2026-09-10T07:17:40Z")
Hi! I tried to use PhyloPhlAn version 3.2.1 (13 February 2026) for MAGs, and saw that output file don’t have two MAGs, they are totally gone I have checked in each folder of tmp. In log file I have 152 as input files, … --- ## [Using CONQUR-corrected matrices in MaAsLin 3](https://forum.biobakery.org/t/using-conqur-corrected-matrices-in-maaslin-3/9051)
**Author:** [@Esther\_Crivelli\_Gali](https://forum.biobakery.org/u/Esther_Crivelli_Gali)\ **Replies:** 1\ **Last updated:** [September 5, 2026, 5:16am UTC](https://forum.biobakery.org/t/using-conqur-corrected-matrices-in-maaslin-3/9051 "2026-09-05T05:16:06Z")
Hi BioBakery Team, We pre-corrected our raw microbiome count matrices for batch effects using CONQUR. We are now analyzing associations with clinical outcomes using MaAsLin 3. To validate our pipeline, should we omit B… --- ## [HUMAnN: Depth-normalization with spike-ins](https://forum.biobakery.org/t/humann-depth-normalization-with-spike-ins/9048)
**Author:** [@paulamli](https://forum.biobakery.org/u/paulamli)\ **Replies:** 3\ **Last updated:** [September 5, 2026, 5:11am UTC](https://forum.biobakery.org/t/humann-depth-normalization-with-spike-ins/9048 "2026-09-05T05:11:26Z")
Hello everyone, We’re using spike-ins (cells added to the sample at a known amount) as the basis for an absolute quantification pipeline on shotgun metagenomics data from stool samples, and using HUMAnN to obtain functi… --- ## [Replotting MaAsLin3 output in specific column order](https://forum.biobakery.org/t/replotting-maaslin3-output-in-specific-column-order/9047)
**Author:** [@franciscodanieldavi](https://forum.biobakery.org/u/franciscodanieldavi)\ **Replies:** 2\ **Last updated:** [September 2, 2026, 5:00pm UTC](https://forum.biobakery.org/t/replotting-maaslin3-output-in-specific-column-order/9047 "2026-09-02T17:00:41Z")
Hi Will, I am running MaAsLin3 using the following code: set.seed(1) fit\_out \<- maaslin3(input\_data = seqtab.nochim, input\_metadata = sample\_data, output = "Ox\_vs\_time", … --- ## [MaAsLin 3 model for paired samples across independent sampling days](https://forum.biobakery.org/t/maaslin-3-model-for-paired-samples-across-independent-sampling-days/9044)
**Author:** [@awu](https://forum.biobakery.org/u/awu)\ **Replies:** 6\ **Last updated:** [August 30, 2026, 7:14pm UTC](https://forum.biobakery.org/t/maaslin-3-model-for-paired-samples-across-independent-sampling-days/9044 "2026-08-30T19:14:10Z")
Hi, I’m new with MaAslin3 and I was trying to figure out what would be the best setting for my analysis but after a lot of reading I’m still not clear. So I decided to ask here hoping that someone can help me. I am ana… --- ## [MetaPhlAn 4 + HUMAnN 4 compatibility](https://forum.biobakery.org/t/metaphlan-4-humann-4-compatibility/8523)
**Author:** [@franzosa](https://forum.biobakery.org/u/franzosa)\ **Replies:** 24\ **Last updated:** [August 30, 2026, 5:15pm UTC](https://forum.biobakery.org/t/metaphlan-4-humann-4-compatibility/8523 "2026-08-30T17:15:15Z")
HUMAnN users - I’m making a quick pinned post about MetaPhlAn 4 + HUMAnN 4 compatibility since we’re seeing a lot of posts raising similar issues. We will follow up with more detailed information in the short term, and l… --- ## [SGB2GTDB v232 utils script](https://forum.biobakery.org/t/sgb2gtdb-v232-utils-script/9043)
**Author:** [@chuiyang-mok](https://forum.biobakery.org/u/chuiyang-mok)\ **Replies:** 0\ **Last updated:** [August 25, 2026, 9:33am UTC](https://forum.biobakery.org/t/sgb2gtdb-v232-utils-script/9043 "2026-08-25T09:33:06Z")
Hi there, I’m writing to try to understand the sourcecode of SGB2GTDB. The latest GTDB database has been updated to version 232, and multiple MAGs-focused metagenomics tools were also updating theirs to v232. I was think… --- ## [Strainphlan sample2markers.py ERROR](https://forum.biobakery.org/t/strainphlan-sample2markers-py-error/8650)
**Author:** [@LEEzhu0110](https://forum.biobakery.org/u/LEEzhu0110)\ **Replies:** 10\ **Last updated:** [August 21, 2026, 3:32pm UTC](https://forum.biobakery.org/t/strainphlan-sample2markers-py-error/8650 "2026-08-21T15:32:06Z")
Dear developers, I met a problem when running strainphlan(under metaphlan version=4.2.4) with command ”sample2markers.py -i vdb3.sam -f sam -o consensus\_markers2 -n 1 -d /mpa\_vOct22\_CHOCOPhlAnSGB\_202403.pkl”.It shows:\[E… --- ## [Impossible to set --max-memory for Trimmomatic](https://forum.biobakery.org/t/impossible-to-set-max-memory-for-trimmomatic/8939)
**Author:** [@RikkeML](https://forum.biobakery.org/u/RikkeML)\ **Replies:** 7\ **Last updated:** [August 21, 2026, 3:31pm UTC](https://forum.biobakery.org/t/impossible-to-set-max-memory-for-trimmomatic/8939 "2026-08-21T15:31:53Z")
Kneaddata v.0.12.4 I am trying to use Kneaddata’s --max-memory flag for Trimmomatic, to avoid Java heap space errors for large fastq files (with threads set to 16, I got this error for a 9GB fastq.gz file). I found tha… --- ## [Inquiry on inferring directionality of HGT events in WAAFLE](https://forum.biobakery.org/t/inquiry-on-inferring-directionality-of-hgt-events-in-waafle/9038)
**Author:** [@Tefero](https://forum.biobakery.org/u/Tefero)\ **Replies:** 0\ **Last updated:** [August 18, 2026, 9:20pm UTC](https://forum.biobakery.org/t/inquiry-on-inferring-directionality-of-hgt-events-in-waafle/9038 "2026-08-18T21:20:26Z")
I am writing to ask how WAAFLE infers the direction of HGT events. WAAFLE assigns directionality to syntenies such as ABA, AB~A, and ABA~A, but not to patterns such as ABABA, ABA\*A, AB\*AA, ABA~\*A, or AB~\*AA. Could you c… --- ## [Analysis taking too much time](https://forum.biobakery.org/t/analysis-taking-too-much-time/9023)
**Author:** [@Rishav\_Sahil](https://forum.biobakery.org/u/Rishav_Sahil)\ **Replies:** 3\ **Last updated:** [August 18, 2026, 12:20pm UTC](https://forum.biobakery.org/t/analysis-taking-too-much-time/9023 "2026-08-18T12:20:31Z")
Hello all, I am using this command phylophlan -i /mnt/bigdata/nikhil/Bradyrhizobium\_genomes -d phylophlan --databases\_folder /mnt/bigdata/nikhil/Bradyrhizobium\_genomes/database --diversity medium --configs\_folder /mnt/b… --- ## [Recommendation for supplementary methods to validate MaAsLin 3 results on MetaPhlAn 4 data](https://forum.biobakery.org/t/recommendation-for-supplementary-methods-to-validate-maaslin-3-results-on-metaphlan-4-data/9037)
**Author:** [@Bluuuec](https://forum.biobakery.org/u/Bluuuec)\ **Replies:** 2\ **Last updated:** [August 17, 2026, 1:01am UTC](https://forum.biobakery.org/t/recommendation-for-supplementary-methods-to-validate-maaslin-3-results-on-metaphlan-4-data/9037 "2026-08-17T01:01:35Z")
Dear MaAsLin 3 team, We are using MaAsLin 3 on MetaPhlAn 4 relative abundance data and identified both prevalence-driven (the majority) and abundance-driven biomarkers. To anticipate potential reviewers’ requests for a… --- ## [How long does it usually take to build a phylogenetic tree with PhyloPhlAn 3 using about 30 genomes?](https://forum.biobakery.org/t/how-long-does-it-usually-take-to-build-a-phylogenetic-tree-with-phylophlan-3-using-about-30-genomes/8714)
**Author:** [@dwenchao234](https://forum.biobakery.org/u/dwenchao234)\ **Replies:** 2\ **Last updated:** [August 16, 2026, 8:41pm UTC](https://forum.biobakery.org/t/how-long-does-it-usually-take-to-build-a-phylogenetic-tree-with-phylophlan-3-using-about-30-genomes/8714 "2026-08-16T20:41:13Z")
This is my first time using PhyloPhlAn 3. I used the command below to build a tree for about 30 genomes, and the program has been running for over six days without finishing. The log files stopped showing any new output … --- ## [Maaslin3 output linear association plots blank](https://forum.biobakery.org/t/maaslin3-output-linear-association-plots-blank/9034)
**Author:** [@franciscodanieldavi](https://forum.biobakery.org/u/franciscodanieldavi)\ **Replies:** 8\ **Last updated:** [August 13, 2026, 10:37pm UTC](https://forum.biobakery.org/t/maaslin3-output-linear-association-plots-blank/9034 "2026-08-13T22:37:58Z")
Hi everyone, I am currently using Maaslin3 tool to perform a DA analysis to determine associations between Oxygen concentrations in oil amended cultures using the following parameters: fit\_out \<- maaslin3(input\_data = … --- ## [Using Melonnpan wiht HUMAnN4](https://forum.biobakery.org/t/using-melonnpan-wiht-humann4/9036)
**Author:** [@Florencia\_Penalba](https://forum.biobakery.org/u/Florencia_Penalba)\ **Replies:** 0\ **Last updated:** [August 13, 2026, 7:57pm UTC](https://forum.biobakery.org/t/using-melonnpan-wiht-humann4/9036 "2026-08-13T19:57:32Z")
I have been using humann v4.0.0.alpha.1 and I have several results. I want to predict the metabolites for this samples. I have tried using melonnpan 0.99.0.predict but it returns an error “No common IDS found between tra… --- ## [Normalisation (CLR, TSS or NONE) for coverM abundance](https://forum.biobakery.org/t/normalisation-clr-tss-or-none-for-coverm-abundance/9035)
**Author:** [@kimndlovu](https://forum.biobakery.org/u/kimndlovu)\ **Replies:** 4\ **Last updated:** [August 13, 2026, 3:22pm UTC](https://forum.biobakery.org/t/normalisation-clr-tss-or-none-for-coverm-abundance/9035 "2026-08-13T15:22:18Z")
Hello MaAsLin3 developers and users, I have metagenomic dataset and the abundance is trimmed mean from coverM that has been normalised by sequencing depth. I have filtered to remove samples with rowSum=0 and taxa with c… --- ## [Absolute cell counts using spike-in in MetaPhlAn](https://forum.biobakery.org/t/absolute-cell-counts-using-spike-in-in-metaphlan/9033)
**Author:** [@Stefano\_Cardinale](https://forum.biobakery.org/u/Stefano_Cardinale)\ **Replies:** 0\ **Last updated:** [August 12, 2026, 3:06pm UTC](https://forum.biobakery.org/t/absolute-cell-counts-using-spike-in-in-metaphlan/9033 "2026-08-12T15:06:30Z")
Hello! I would like to ask a question about the use of spiked- in species with MetaPhlAn. We used samples with spiked species in our analysis, is there support in MetaPhlAn to use spiked species to get absolute cell coun… --- ## [MetaPhlAn and GPU - BWA-mem](https://forum.biobakery.org/t/metaphlan-and-gpu-bwa-mem/9032)
**Author:** [@Stefano\_Cardinale](https://forum.biobakery.org/u/Stefano_Cardinale)\ **Replies:** 0\ **Last updated:** [August 12, 2026, 2:57pm UTC](https://forum.biobakery.org/t/metaphlan-and-gpu-bwa-mem/9032 "2026-08-12T14:57:27Z")
Hello, I was wondering whether it is possible to use a different aligner’s SAM output with MetaPhlAn now. For example, in my group we would like to use some versions of aligners (like BWA) that can run on GPU, has anyone… --- ## [CAMEO - CAZymes with HUMAnN](https://forum.biobakery.org/t/cameo-cazymes-with-humann/9030)
**Author:** [@koitaxoumemesa](https://forum.biobakery.org/u/koitaxoumemesa)\ **Replies:** 0\ **Last updated:** [August 11, 2026, 8:05pm UTC](https://forum.biobakery.org/t/cameo-cazymes-with-humann/9030 "2026-08-11T20:05:53Z")
Hi everyone, @Matthew\_Bolino put together a handy mapping file for CAZyme regrouping with HUMAnN, CAMEO. It’s now published in Microbiology Resource Announcements and the GitHub with more info is linked below. This sho… --- ## [Negative Values in GTDB Converted MetaPhlAn4 Result File](https://forum.biobakery.org/t/negative-values-in-gtdb-converted-metaphlan4-result-file/9017)
**Author:** [@aegreen32](https://forum.biobakery.org/u/aegreen32)\ **Replies:** 5\ **Last updated:** [August 10, 2026, 2:43pm UTC](https://forum.biobakery.org/t/negative-values-in-gtdb-converted-metaphlan4-result-file/9017 "2026-08-10T14:43:18Z")
Hello! I recently used the newest version of the sgb\_to\_gtdb\_profile.py script with the GTDB r226 updated mapping file, and as I was starting to analyze the results I realized that the conversion script added negative v… --- ## [Maaslin3 significant feature not shown in summary plot](https://forum.biobakery.org/t/maaslin3-significant-feature-not-shown-in-summary-plot/7782)
**Author:** [@dlepp](https://forum.biobakery.org/u/dlepp)\ **Replies:** 5\ **Last updated:** [August 8, 2026, 12:33am UTC](https://forum.biobakery.org/t/maaslin3-significant-feature-not-shown-in-summary-plot/7782 "2026-08-08T00:33:49Z")
I am using maaslin3 v‘0.99.1’ in Rstudio, and am having an issue where a feature is present in the significant\_results.tsv file with no model errors, but is not showing up on the summary plot. I have imported the ggplot … --- ## [Issue with baqlava-db download](https://forum.biobakery.org/t/issue-with-baqlava-db-download/9026)
**Author:** [@eodonne3](https://forum.biobakery.org/u/eodonne3)\ **Replies:** 1\ **Last updated:** [August 6, 2026, 9:22pm UTC](https://forum.biobakery.org/t/issue-with-baqlava-db-download/9026 "2026-08-06T21:22:15Z")
Hi, I am attempting to download the BAQLaVa reference database from: https://huttenhower.sph.harvard.edu/baqlava-db/databases.V0.5.tar.gz The URL redirects to: https://g-227ca.190ebd.75bc.data.globus.org/baqlava-db/d… --- ## [Turn off fitlering in anpan\_repeated\_measures](https://forum.biobakery.org/t/turn-off-fitlering-in-anpan-repeated-measures/9024)
**Author:** [@rob](https://forum.biobakery.org/u/rob)\ **Replies:** 4\ **Last updated:** [August 6, 2026, 8:57pm UTC](https://forum.biobakery.org/t/turn-off-fitlering-in-anpan-repeated-measures/9024 "2026-08-06T20:57:20Z")
I’m using anpan\_repeated\_measures on presence-absence data with multiple samples per individual. I’ve run the profiles through other filtering pipelines, so I’ve turned anpan filtering off with filtering\_method = “none”,… --- ## [StrainPhlAn Reference Genomes](https://forum.biobakery.org/t/strainphlan-reference-genomes/8556)
**Author:** [@silvtal](https://forum.biobakery.org/u/silvtal)\ **Replies:** 1\ **Last updated:** [August 4, 2026, 10:26am UTC](https://forum.biobakery.org/t/strainphlan-reference-genomes/8556 "2026-08-04T10:26:13Z")
Hello BioBakery community, I’m currently trying to run a strain-level analysis using StrainPhlAn 4.0 on a large metagenomic dataset of mouse gut samples, previously processed with KneadData and MetaPhlAn (ChocoPhlAn Jun… [Next page](https://forum.biobakery.org/latest.md?page=1)