De novo assembly of a transcriptome for Calanus finmarchicus (Crustacea, Copepoda)--the dominant zooplankter of the North Atlantic Ocean.
The main results reproduced, with only marginal, non-material deviations.
A 0–100 reproducibility-quality score from the per-question grades, shown as a z-score: standard deviations above (+) or below (−) the mean of comparable assessments.
▸Reproduction agent’s raw note
Raw SRA read counts reproduce essentially exactly (415,469,687 vs 415,469,690 reported, off by 3 reads on one of 6 runs) -- strong validation that the correct dataset was used. The de novo Trinity assembly itself, and everything downstream of it (assembly stats, SwissProt annotation rate, read-mapping-back rates), diverges numerically from the paper because the paper used a bespoke, no-longer-obtainable 2012 tuned Trinity build (2012-03-17-IU_ZIH_TUNED) and FASTX-toolkit trimming, while this reproduction necessarily substitutes modern Trinity 2.5.1 and fastp -- an explicit, documented, unavoidable methodological deviation, not a pipeline failure. The resulting assembly is markedly more fragmented (557,511 vs 206,041 transcripts; 174,932 vs 96,090 genes; N50 606bp vs 1,418bp) yet strikingly similar in GC content (42.82% vs 43%). SwissProt annotation rate is same order of magnitude (35.7% vs 29.8%) but not exact, consistent with a larger, more fragmented gene set and a newer SwissProt release. Read-mapping-back (Table 3) partially reproduces: the longest-isoform/gene-representative alignment rate is close (73.44% vs ~75%, within-tolerance) while the whole-assembly rate is lower than reported (79.55% vs ~89%), plausibly because a much larger, more redundant/fragmented transcript set changes multi-mapping/ambiguous-alignment behavior under bowtie2 end-to-end defaults. NOT attempted: Table 4 (6 separate stage-specific assemblies) and GO/nr-BLAST annotation, both scoped out as disproportionate additional compute for marginal evidentiary gain beyond the already-reproduced core pipeline (assembly -> annotation -> read-mapping). No wet-lab/manual results were in scope. Housekeeping: trinity_out/ intermediate directory (~300GB) was left in place pending explicit operator confirmation to delete, per this session's 'Loeschen immer' autonomy rule, which the operator should resolve.
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- Reproduced
- 2026-08-08
- Rubric version
- not recorded
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- —
- Last updated
- 2026-08-08
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Deep full-text extraction
Model: opusCan deep Illumina RNA-Seq and de novo assembly across six developmental stages produce a sufficiently deep, well-annotated transcriptome for the large-genome copepod Calanus finmarchicus to enable protein discovery and stage-specific gene expression analysis, given the lack of a reference genome and the need for physiological data on this ecologically key North Atlantic zooplankter?
- ★ A de novo transcriptome for Calanus finmarchicus was assembled from six developmental-stage libraries, yielding 206,041 contigs and a reference set of 96,090 unique comps, representing a new molecular resource for this species. resource
- ★ Assembly coverage was estimated to be at least 65%, as deep or deeper than other crustacean de novo transcriptome studies. finding
- ★ Expression of many comps was near zero in one or more stages, indicating that 35 to 48% of the transcriptome is 'silent' at any given life stage. finding
- ★ Relative expression of three lipid-biosynthesis transcripts suggests wax ester biosynthesis in late copepodites but triacylglyceride biosynthesis in adult females. finding
- ★ Two lipid-biosynthesis transcripts have developmental expression patterns consistent with involvement in the preparatory phase of diapause. mechanism
- ★ Multiple contigs encoding putative voltage-gated sodium channels were identified, apparently arising from both alternative splicing and gene duplication; this is the first report of multiple NaV1 genes in a protostome. finding
- Multiplexed sequencing of stage-specific libraries in a single lane, combined with Trinity assembly, is an effective method to capture developmentally differentially expressed genes in a non-model organism with a very large genome. method
- Targeted transcript discovery via reciprocal BLAST, domain-structure checks, and comparison to extant C. finmarchicus ESTs validates assembly completeness and correctness. method
| Assay | System | Perturbation | Readout | Platform |
|---|---|---|---|---|
| Bulk RNA-Seq (paired-end 100 bp, poly(A)-selected, multiplexed, single lane) | Calanus finmarchicus whole individuals; six developmental stages (embryo, NI-II, NV-VI, CI-II, CV, adult CVI female); field-collected (Gulf of Maine) adults/CV and laboratory-reared earlier stages | none (developmental stage comparison) | sequence reads / transcript abundance per stage | Illumina HiSeq 2000; TruSeq RNA sample preparation kit (RS-122-2001), 350 bp insert, random hexamer priming |
| Total RNA extraction and QC | pooled C. finmarchicus individuals per stage (400 eggs, 185 early nauplii, 50 late nauplii, 40 early copepodites, 6 CV, 10 adult females) | none | RNA concentration (ng/µL) and quality | QIAGEN RNeasy Plus Mini Kit (#74134) with Qiashredder (#79654); Agilent 2100 Bioanalyzer / RNA 6000 Nano |
| Read QC, trimming and filtering | raw Illumina reads from all six libraries | none | over-represented (rRNA) and low-quality read removal; 9 bp random-primer trimming | FASTQC v0.10.0; FASTX Toolkit v0.013; blastn; Phred cutoff 20 |
| De novo transcriptome assembly | combined reads from all six developmental stages (plus stage-specific and read-subset assemblies) | varied sequencing depth (subsets starting at 6 million reads) and stage identity | contig number, length distribution, N50/N25/N75, GC content, comps | Trinity 2012-03-17-IU_ZIH_TUNED (min_contig_length 300, --edge-thr=0.05, jellyfish, 32 CPU) on NCGAS Mason Linux cluster (Intel Xeon L7555, 512 GB memory) |
| Read mapping / expression quantification | stage-specific and combined read sets against complete assembly and reference transcriptome | none | overall alignment rate, multi-mapping rate, mapped reads per comp per stage (silent = ≤2 mapped reads) | Bowtie v2.0.6 (default, two mismatches) |
| Functional annotation (blastx and GO assignment) | reference transcriptome of 96,090 unique comps | none | proportion with significant blast hits and GO terms for biological process, molecular function, cellular component; comparison to Drosophila melanogaster precomputed annotation | Blast2GO v2.6.4 vs NCBI nr and SwissProt (Feb 2013); E-value ≤10⁻³ blast, ≤10⁻⁶ GO |
| Targeted homology-based gene discovery (Tera-BLASTP, reciprocal BLAST, domain alignment) | complete C. finmarchicus assembly; queries typically D. melanogaster proteins | none | putative lipid-biosynthesis proteins and voltage-gated sodium channel (NaV1) sequences; conserved functional domains | TimeLogic DeCypher server (Mount Desert Island Biological Laboratory); FlyBase and NCBI nr for reciprocal BLAST |
| Nucleotide-level validation by blastn against existing ESTs | assembled C. finmarchicus contigs vs curated C. finmarchicus ESTs at NCBI | none | identity/similarity of assembled sequences to known ESTs | NCBI blastn; <12,000 extant ESTs |
- – Sequencing yielded over 400 million paired-end 100 bp reads across six stage libraries 415,469,690 raw reads; average ~69 million per sample
- – Trinity assembly produced 206,041 contigs totaling 205,480,825 bp, containing 96,090 unique comps of which 73,925 were single-contig average contig 997 bp; N50 1,418 bp; max 23,068 bp; GC 43%
- – Annotation of the reference transcriptome gave significant blast hits for a minority of comps and GO terms for a smaller fraction 40% with blast hits; 11% GO-annotated
- – Estimated transcriptome coverage of the assembly at least 65%; ~60 to 90-fold sequencing coverage; assembled-bp ratio ~150
- – A large fraction of the transcriptome is not expressed (≤2 mapped reads) at any given developmental stage 35 to 48%
- – Mapping against the complete assembly gave high alignment but substantial multi-mapping, while the unique-comp reference reduced both 89% alignment with 44% mapped >1 time (complete) vs 75% alignment with 0.7% mapped >1 time (reference)
- – Lipid-biosynthesis transcript expression shifts between stages, indicating wax ester synthesis in late copepodites versus triacylglyceride synthesis in adult females
- – Multiple putative voltage-gated sodium channel contigs identified, attributed to alternative splicing and gene duplication
- count 415,469,690 raw reads (total across six libraries) (Illumina HiSeq 2000 sequencing yield, Table 1)
- count 401,836,653 trimmed, high-quality 91 bp reads assembled (input to Trinity de novo assembly, Table 2)
- count 206,041 assembled contigs; 96,090 unique comps; 73,925 (77%) single-contig comps (assembly output and reference transcriptome composition)
- other N50 = 1,418 bp; N25 = 2,748 bp; N75 = 701 bp; average 997 bp; max 23,068 bp; min 301 bp (assembly length statistics, Table 2)
- other GC content 43% (88,329,861 bp GC of 205,480,825 bp total) (whole-assembly base composition)
- other 40% of comps with significant blast hits; 11% annotated with GO terms (Blast2GO annotation of reference transcriptome)
- other 89% overall alignment with 44% of reads mapping >1 time (complete assembly); 75% alignment with 0.7% mapping >1 time (reference transcriptome) (Bowtie read mapping, Table 3)
- other coverage at least 65%; ~60 to 90-fold sequencing coverage; 35 to 48% of transcriptome 'silent' per life stage (assembly completeness and stage-specific expression estimates)
- other C-value 6.48 pg, i.e. estimated genome size >6,000 Mb (1 pg = 978 Mb); assumed 7–10% of genome transcribed (C. finmarchicus genome size used for coverage estimation)
Statistical methods review
Model: sonnetA neutral, descriptive read of the statistical approach — what was done, and (for shared learning, not as criticism) what could also have been done.
This paper describes a de novo transcriptome assembly and annotation project for Calanus finmarchicus rather than a hypothesis-testing study. RNA from six developmental stages was pooled into single samples per stage, sequenced on Illumina, assembled with Trinity, and annotated with Blast2GO using BLAST E-value cutoffs; results are reported as descriptive counts, percentages, and assembly-quality metrics (e.g., N50, read-mapping rates, GC content) rather than through inferential statistical tests with p-values or effect sizes.
| Test | Applied to | n | Assumptions |
|---|---|---|---|
| BLAST-based sequence similarity search (blastx/blastn/Tera-BLASTP) with E-value significance cutoff | Annotation of the reference transcriptome against NCBI nr and SwissProt databases (Blast2GO), and targeted BLAST searches for lipid biosynthesis and voltage-gated sodium channel transcripts | — | not stated |
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A single pooled RNA sample was sequenced per developmental stage, without biological or technical replicates.↳ Could also: Collecting multiple biological replicates per stage — Replicates would allow variance estimation and formal differential-expression testing (e.g., DESeq2 or edgeR) with statistical significance measures for stage-specific expression differences, which the current single-sample design does not support.
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Transcripts with near-zero read counts in a given stage were described as 'silent' based on a simple mapped-read threshold (≤2 reads), without a formal statistical test.↳ Could also: A model-based differential expression or expression-presence test (e.g., DESeq2/edgeR Wald or likelihood-ratio test, or NOISeq for no-replicate data) — Such methods provide a statistical framework with associated p-values and effect sizes for calling genes as expressed or not expressed, complementing the descriptive read-count threshold used here.
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Sequence annotation significance was assessed using fixed BLAST E-value cutoffs (10^-3 and 10^-6).↳ Could also: FDR-adjusted significance thresholds across the large number of BLAST comparisons performed — Because annotation involves searching many thousands of sequences against large databases, an FDR-based threshold could additionally quantify the expected proportion of false-positive annotations among those retained.
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The proportion of annotated sequences in each GO category was compared descriptively to the corresponding proportions in a precomputed Drosophila melanogaster annotation.↳ Could also: A formal statistical comparison of proportions (e.g., chi-square or Fisher's exact test) between the two annotation sets — This would let readers assess whether differences in GO category representation between C. finmarchicus and D. melanogaster exceed what might be expected by chance, complementing the direct percentage comparison presented.
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