Decontamination, pooling and dereplication of the 678 samples of the Marine Microbial Eukaryote Transcriptome Sequencing Project
Open accessAbstract
Objectives: Complex algae are photosynthetic organisms resulting from eukaryote-to-eukaryote endosymbiotic-like interactions. Yet the specific lineages and mechanisms are still under debate. That is why large scale phylogenomic studies are needed. Whereas available proteomes provide a limited diversity of complex algae, MMETSP (Marine Microbial Eukaryote Transcriptome Sequencing Project) transcriptomes represent a valuable resource for phylogenomic analyses, owing to their broad and rich taxonomic sampling, especially of photosynthetic species. Unfortunately, this sampling is unbalanced and sometimes highly redundant. Moreover, we observed contaminated sequences in some samples. In such a context, tree inference and readability are impaired. Consequently, the aim of the data processing reported here is to release a unique set of clean and non-redundant transcriptomes produced through an original protocol featuring decontamination, pooling and dereplication steps. Data description: We submitted 678 MMETSP re-assembly samples to our parallel consolidation pipeline. Hence, we combined 423 samples into 110 consolidated transcriptomes, after the systematic removal of the most contaminated samples (186). This approach resulted in a total of 224 high-quality transcriptomes, easy to use and suitable to compute less contaminated, less redundant and more balanced phylogenies.
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Written from the paper’s full text.
TL;DR
The authors processed 678 MMETSP re-assembly samples through a pipeline of decontamination, pooling, and dereplication to produce a cleaner, less redundant transcriptome resource. The final release contains 224 high-quality transcriptomes, including 172 photosynthetic and 145 complex algal samples.
Key findings
- From 678 MMETSP re-assembly samples, 16 were discarded initially (14 with fewer than 5,000 sequences, 1 corrupted FASTQ, 1 unknown organism), leaving 662.
- Screening with Forty-Two using 78 ribosomal protein markers discarded 89 samples as too contaminated, leaving 573; pooling combined 423 candidate samples into 110 consolidated transcriptomes, with 26 additional uncombined singletons, aggregating 313 redundant samples.
- After further removal of 36 transcriptomes corresponding to 97 original samples, the final dataset contained 224 transcriptomes, of which 172/224 were photosynthetic and 145/224 were complex algae.
- A GAPDH phylogeny comparison indicated the new dataset was less contaminated, less redundant, and more taxonomically balanced than using all 570 plastid-bearing samples.
Methods
- The study used the 678 MMETSP re-assembled transcriptomes as input and applied a consolidation pipeline featuring decontamination, pooling, and dereplication.
- Contamination was assessed with Forty-Two using 78 ribosomal protein markers and with CroCo/Sobek for cross-contaminations; samples sharing a cross-contamination value ≥10% were combined, and CD-HIT-EST at 95% sequence identity was used to remove intra-sample redundancy before further contamination screening.
- Completeness was assessed with BUSCO, and usefulness was illustrated with GAPDH phylogenies comparing 570 plastid-bearing samples against the 172 transcriptomes from the pipeline.
Limitations
- The paper states that combining some samples at the genus level may lose resolution at the species/strain level, and that sequence redundancy remains substantial, especially among dinoflagellates.
- The paper states that contaminations remain in the cleaned transcriptomes; ciliates are noted as highly contaminated, requiring a phylum-based outlier approach because a global threshold was impracticable.
- The paper states that foreign-sequence contamination can be a “necessary evil” because transferred genes in a plastid-acquisition context would be undetectable in a completely uncontaminated transcriptome.
Model-written summary. Check it against the paper before relying on it.
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Cites
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Claims the paper makes
0 of 9 cited sources checked · 0 supported · 0 flagged
- Findingown data
Both detection methods combined pinpointed 36 transcriptomes, corresponding to 97 original samples.
“Both detection methods combined pinpointed 36 transcriptomes, corresponding to 97 original samples (Data file 6, Data file 7, Data file 1).”
Quote in the paperNot a substantial claim - Findingreasoning
Contaminations remain in the provided set of cleaned transcriptomes.
“Similarly, even if we provide a set of cleaned transcriptomes, contaminations remain.”
Quote in the paperNot a substantial claim - Findingown data
Contaminated sequences were observed in some MMETSP samples.
“Moreover, we observed contaminated sequences in some samples.”
- Also stated in: § Data description · plain
“we observe high redundancy and contaminations”
Quote in the paperSubstantial claim - Also stated in: § Data description · plain
- Findingreasoning
Even though combined samples were dereplicated, sequence redundancy remains substantial, especially among dinoflagellates.
“Even though combined samples were dereplicated, sequence redundancy remains substantial, especially among dinoflagellates, which are known for bearing multiple copies of their genes.”
Quote in the paperNot a substantial claim - Findingown data
186 most contaminated samples were systematically removed.
“after the systematic removal of the most contaminated samples (186)”
Quote in the paperSubstantial claim
- Premisecitation
MMETSP transcriptomes represent a valuable resource for phylogenomic analyses, owing to their broad and rich taxonomic sampling, especially of photosynthetic species.
“MMETSP (Marine Microbial Eukaryote Transcriptome Sequencing Project) transcriptomes represent a valuable resource for phylogenomic analyses, owing to their broad and rich taxonomic sampling, especially of photosynthetic species.”
- Also stated in: § Objective · plain
“richly sampled MMETSP transcriptomes [9, 10]”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [9] Keeling PJ, et al. The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol. 2014;12(6):e1001889. source
Not checked in the free run.
- cites [10] Johnson LK, et al. Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes. Gigascience. 2019. https://doi.org/10.1093/gigascience/giy158. source
Not checked in the free run.
- Also stated in: § Objective · plain
- Interpretationown data
The enrichment allows more robust evolutionary interpretations.
“thereby allowing more robust evolutionary interpretations”
Quote in the paperSubstantial claim - Interpretationown data
The data set is suitable to compute less contaminated, less redundant and more balanced phylogenies.
“suitable to compute less contaminated, less redundant and more balanced phylogenies”
Quote in the paperSubstantial claim - Interpretationreasoning
Contamination by foreign sequences is a necessary evil for studying transferred genes in a plastid acquisition context, because those genes would be undetectable in a completely uncontaminated transcriptome.
“Second, in other cases, contamination by foreign sequences is a “necessary evil” for the reason that we plan to study transferred genes in a plastid acquisition context, and those would be undetectable in a completely “uncontaminated” transcriptome.”
Quote in the paperSubstantial claim - Interpretationreasoning
Removing the most contaminated samples instead of decontaminating them allows more reliable interpretation of downstream phylogenetic analyses at the expense of a moderate loss of taxonomic breadth and richness.
“This somewhat radical method has the advantage of allowing a more reliable interpretation of downstream phylogenetic analyses at the expense of a moderate loss of taxonomic breadth and richness.”
Quote in the paperSubstantial claim - Interpretationreasoning
Combining some samples at the genus level might lead to a loss of resolution at the species/strain level within a given genus.
“The way some samples were combined at the genus level might lead to a loss of resolution at the species/strain level within a given genus.”
Quote in the paperNot a substantial claim
- May combine two claims. 12
- Classified as probably not a substantial claim. 1
May combine two claims.
Findingown dataComparison of GAPDH trees shows that the enrichment performed with the data set described here is less contaminated, less redundant and more balanced from a taxonomic point of view.
“The comparison of these two trees shows that the enrichment performed with the data set described here is less contaminated, less redundant and more balanced from a taxonomic point of view, thereby allowing more robust evolutionary interpretations.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThe dual strategy yielded 110 combined samples (out of 423 candidate samples) and 26 uncombined samples, aggregating 313 redundant samples.
“This dual strategy yielded 110 combined samples (out of 423 candidate samples) and 26 uncombined samples (additional singletons), thereby aggregating 313 redundant samples (Data file 1, Data file 3).”
Quote in the paperSubstantial claimMay combine two claims.
Findingnone given124 samples represented unique genera and were not combined.
“However, 124 samples represented unique genera and were not combined.”
Quote in the paperNot a substantial claimMay combine two claims.
Findingnone givenMMETSP sampling is unbalanced and sometimes highly redundant.
“Unfortunately, this sampling is unbalanced and sometimes highly redundant.”
- Also stated in: § Data description · plain
“we observe high redundancy and contaminations”
Quote in the paperSubstantial claim- Also stated in: § Data description · plain
May combine two claims.
Findingown dataOf the remaining 662 samples, 89 were discarded as too contaminated after screening with Forty-Two using 78 ribosomal protein markers, leaving 573 samples.
“The remaining 662 were screened for contaminations with Forty-Two [12, 13] using 78 ribosomal protein markers, and 89 samples were further discarded because they were too contaminated (Data file 1, Data set 1), leaving 573 samples.”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [12] Simion P, et al. A large and consistent phylogenomic dataset supports sponges as the sister group to all other animals. Curr Biol. 2017;27(7):958–67. source
Not checked in the free run.
- cites [13] Irisarri I, et al. Phylotranscriptomic consolidation of the jawed vertebrate timetree. Nat Ecol Evol. 2017;1(9):1370–8. source
Not checked in the free run.
May combine two claims.
Findingown dataThe combination reduced the number of samples by more than 50% for the majority of main phyla, resulting in a total of 260 transcriptomes.
“From a taxonomic point of view, this combination had the effect of reducing the number of samples by more than 50% for the majority of main phyla (Data file 4), resulting in a total of 260 transcriptomes (combined and singletons, Data set 2).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataOut of 678 MMETSP re-assembly samples, 16 were discarded from the start (14 with less than 5000 sequences, 1 with a corrupted FASTQ file, and 1 because the organism was unknown).
“out of the 678 samples in MMETSP re-assemblies, 16 were discarded from the start (14 with less than 5000 sequences, 1 with a corrupted FASTQ file and another one because the organism was unknown).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataThe resulting data set of 224 transcriptomes spans a wide range of organisms, especially among photosynthetic species (172/224), with a majority of complex algae (145/224).
“The resulting data set of 224 transcriptomes (Data set 2) spans a wide range of organisms, especially among photosynthetic species (172/224), with a majority of complex algae (145/224) (Data set 4).”
- Also stated in: § Data description · plain
“This approach resulted in a total of 224 high-quality transcriptomes, easy to use and suitable to compute less contaminated, less redundant and more balanced phylogenies.”
Quote in the paperNot a substantial claim- Also stated in: § Data description · plain
May combine two claims.
InterpretationreasoningContamination and redundancy impair tree inference and readability.
“In such a context, tree inference and readability are impaired.”
- Also stated in: § Data description · plain
“which complicates the study of plastid spread across eukaryotic lineages”
Quote in the paperSubstantial claim- Also stated in: § Data description · plain
Classified as probably not a substantial claim. May combine two claims.
PremisecitationCASH lineages have plastids related to red algae, but the mechanisms by which they were acquired and the ancestors involved remain unclear.
“CASH lineages have plastids related to red algae, but the mechanisms by which they were acquired and the ancestors involved remain unclear [1–3].”
Quote in the paperSubstantial claim0 of 3 sources checked- cites [1] Zimorski V, et al. Endosymbiotic theory for organelle origins. Curr Opin Microbiol. 2014;22:38–48. source
Not checked in the free run.
- cites [2] Ponce-Toledo RI, et al. Horizontal and endosymbiotic gene transfer in early plastid evolution. New Phytol. 2019;224(2):618–24. source
Not checked in the free run.
- cites [3] Sibbald SJ, Archibald JM. Genomic insights into plastid evolution. Genome Biol Evol. 2020;12:978–90. source
Not checked in the free run.
May combine two claims.
PremisecitationMMETSP transcriptomic samples cover a wide range of primary and complex algae and were recently re-assembled.
“MMETSP transcriptomic samples [9] cover a wide range of primary and complex algae and were recently re-assembled [10].”
Quote in the paperSubstantial claim0 of 2 sources checked- cites [9] Keeling PJ, et al. The Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP): illuminating the functional diversity of eukaryotic life in the oceans through transcriptome sequencing. PLoS Biol. 2014;12(6):e1001889. source
Not checked in the free run.
- cites [10] Johnson LK, et al. Re-assembly, quality evaluation, and annotation of 678 microbial eukaryotic reference transcriptomes. Gigascience. 2019. https://doi.org/10.1093/gigascience/giy158. source
Not checked in the free run.
May combine two claims.
PremisereasoningTransferred genes are hardly distinguishable from actual contaminations, prompting the use of clean data to avoid false positives.
“In addition, the inherent nature of those genes, which are transferred from one lineage to another, makes them hardly distinguishable from actual contaminations, prompting the use of data as clean as possible to avoid false positives.”
Quote in the paperSubstantial claim
The paper relies on this without support.
In some cases, such as ciliates, all members of the phylum are highly contaminated.
“First, in some cases, such as ciliates, it is a “feature” of the phylum that all its members are highly contaminated.”
The paper relies on this without support.
Available proteomes provide a limited diversity of complex algae.
“Whereas available proteomes provide a limited diversity of complex algae”
“To overcome the limited diversity of complex algal proteomes”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
Decontamination, pooling, and dereplication methods have been employed to refine transcriptome sequences, rendering them more amenable to analysis with lower contamination, redundancy, and improved balance in phylogenies.
“Subsequent to sequencing, decontamination, pooling, and dereplication methods have been employed to refine these sequences, rendering them more amenable to analysis, with lower levels of contamination, redundancy, and improved balance in the resulting phylogenies [230].”
From Microalgae biofuels: illuminating the path to a sustainable future amidst challenges and opportunities (2024) · cites this paper as [230]
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Self-citationData file 3 - Sobek analysis summary
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Self-citationData set 3 - Forty-Two reports and configuration files (260 transcriptomes)
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Self-citationData set 1 - Forty-Two reports and configuration files (662 individual samples)
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Data set 5 - GAPDH phylogenies
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Self-citationData file 2 - Redundancy drop analysis
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Self-citationData file 1 - Consolidation table
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Data file 6 - Completeness analysis
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Self-citationData file 1 - Sample consolidation report
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Self-citationData set 4 - Taxonomic samplings
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Self-citationData file 4 - Consolidated samples purity (cross-contaminations)
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Self-citationData file 5 - Consolidated samples purity (contaminations)
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- Authors
- Van Vlierberghe, M., Di Franco, A., Philippe, H., & Baurain, D.
- Affiliations
- Centre National de la Recherche Scientifique (FR)
- University of Liège (BE)
- Corresponding author
- Baurain, Denis
- Venue
- BMC Research Notes
- Published
- 2021-08-09
- Volume
- 14
- Issue
- 1
- Pages
- 306-306
- ISSN
- 1756-0500 (ISSN-L)
- PMC
- PMC8353744
- OpenAlex
- W3192110718
- Semantic Scholar
- 7aa8756123daf2165fe4973a65345bc792db3807
- Cited by
- 27
- References
- 34
- Citations by year
- 2026: 2 · 2025: 6 · 2024: 4 · 2023: 4 · 2022: 6 · 2021: 5
- FWCI
- 1.49
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- BMC Research Notes (publishedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- HAL (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- ORBi (University of Liège) (submittedVersion, other-oa)
- Funders
- Fonds pour la Formation à la Recherche dans l’Industrie et dans l’Agriculture
- Université de Liège (“Crédit de démarrage 2012” SFRD-12/04)
- Fonds De La Recherche Scientifique - FNRS (“Crédit de recherche 2014” CDR J.0080.15)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protist diversity and phylogeny
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Physical Sciences › Environmental Science › Ecology › Environmental DNA in Biodiversity Studies
- Keywords
- sample pooling, dereplication, phylogenomics, endosymbiosis, photosynthetic eukaryotes, transcriptome assembly, sequencing contamination, phylogenetic inference
- MeSH terms
- Decontamination, Phylogeny, Plants, Eukaryota, Transcriptome
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
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