--- slug: the-microbiome-in-the-blank passed: 2026-11-02 by: ccr-81332fb3-8nrbtu wave, one independent agent on oversight/claims-pass.md (pre-deploy; reader check on local copies with AW_DIR; every source-record entry read against its kept copy and live source) checked: 126 confirmed: 88 wrong: 0 unverifiable: 0 observed: 38 stale: 0 --- # Claims pass: The Microbiome in the Blank Live page: not deployed yet; step 2 skipped. Reader check (pre-deploy, local copies): `AW_DIR= node verify-the-microbiome-in-the-blank.mjs`, run from a second empty directory holding only the verifier: exit 0, 221 passed, 0 failed (293 passed inside the repository, where it also reads the kept copies and the frontmatter). Step 6: in a scratch copy, "In 27 of the 28 tubes" changed to "In 25 of the 28 tubes" in its sentence, nothing else touched: exit 1, 219 passed, 2 failed (that sentence, and the residual sweep). With one block of each aw-* kind (aw-plain as the real meta placard), the Cloudflare beacon, the pulse script and /assay/record.js injected, the copy still passes (exit 0, 222 passed), and a planted "24" in a sentence of it still fails (exit 1). `--deep` from the same empty directory: first run exit 1 (ENA answered 503 for two of the six downloads), second run exit 0, 233 passed, 0 failed. Mutation sweep: the-microbiome-in-the-blank: 402 mutated, 402 caught, 0 survived (text 0, meta 0, year 0). `assay/sources.mjs the-microbiome-in-the-blank --online`: 120 entries, offline sound; 76 FOUND, 3 UNREADABLE, 41 in the repository, 6 DOI MATCH. The 3 UNREADABLE (lauder-psp-kit, table-s3, salter-40) are Europe PMC supplementaryFiles zips: I downloaded both zips on 2026-11-02 and read 40168_2016_172_MOESM5_ESM.doc and 12915_2014_87_MOESM1_ESM.docx as text; every excerpt is there, and the kept copies match them (the Salter copy differs only in curly versus straight quote marks). The 41 in the repository were read against the repository and made again: research/the-microbiome-in-the-blank/build.mjs, rerun into a scratch directory from the 84 raw FASTQs (each md5-equal to ENA's live fastq_md5), reproduced lauder.json, salter.json and refs.json byte for byte (git status of the layer unchanged), and the main figures were recomputed a third time in my own Python. The run report, BioSample records, RefSeq records and both NCBI searches were fetched live and match what the page ships. Every quotation (27 passages in 30 quoted spans) was matched to the live Europe PMC text with inline tags removed; the only differences are no-break and thin spaces the page writes as plain spaces. Figure 2b was viewed from the live zip. The Sources link https://europepmc.org/article/PMC/PMC4917942 answered 403 to curl; the REST full text it mirrors reads normally, and I did not test the link as a browser would. Not a finding. ## Claims - CONFIRMED | In a 2016 study of six placentas | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | Discussion: "Here, we compared bacterial DNA recovered from placental samples from six uncomplicated deliveries at term (both fetal side and maternal side internal tissues) to contamination controls." Crossref: Microbiome 4, article 29, issued 2016-06-23 - CONFIRMED | In 2016 Abigail Lauder and colleagues | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | author list opens "Lauder Abigail P."; Crossref 10.1186/s40168-016-0172-3: Abigail P. Lauder first of 10 authors, issued 2016 - CONFIRMED | at the Perelman School of Medicine at the University of Pennsylvania | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | affiliations: "Department of Microbiology, Perelman School of Medicine at the University of Pennsylvania" and "Department of Obstetrics and Gynecology ... Perelman School of Medicine at the University of Pennsylvania" - CONFIRMED | looked for bacterial DNA inside placentas from six uncomplicated deliveries at term, beside control tubes that held no tissue | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "These placental samples were compared to blank swabs waved in air in the clinical laboratory where placental samples were processed, unused sterile swabs, and DNA purifications with no added starting material."; samples "isolated as 0.5 x 0.5 x 0.5 cm cuboidal sections from internal structures within the placenta" - CONFIRMED | Biopsies from the fetal and the maternal side of each placenta were extracted with each of two DNA extraction kits | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | Methods: "One maternal side biopsy sample and one fetal side biopsy sample were selected randomly for DNA purification by both of the methods for each of the six participants in the current study." - CONFIRMED | each biopsy went whole into one kit | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | Method 1 (PSP): "The entirety of each tissue biopsy was placed into a tared bead tube and weighed (samples ranged from 0.11 to 0.48 g)."; Method 2 says the same with "0.12 to 0.66 g" - CONFIRMED | so the two kits worked on different pieces of the same side | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | Method 2: "DNA was isolated from replicate biopsies, saliva, and control samples using the MO BIO PowerSoil DNA Isolation Kit"; collection: "three from the maternal side of the placenta (basal plate), two from the fetal side"; the two kits weighed different ranges, and BioSample gives each kit its own placental sample (FSB.55 vs FetalSideBiopsy55...). The collection sentence alone is ambiguous; "entirety" in both methods plus "replicate biopsies" supports the page - CONFIRMED | the MO BIO PowerSoil kit | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "Two different kits were compared, the STRATEC PSP Spin Stool DNA Plus Kit (henceforth "PSP") and the MO BIO PowerSoil DNA Isolation Kit (abbreviated "MO BIO")" - CONFIRMED | the PSP Spin Stool DNA Plus kit | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/supplementaryFiles | UNREADABLE to the tool (zip); read from the live zip 2026-11-02, 40168_2016_172_MOESM5_ESM.doc as text: "PSP is the abbreviated term used for the STRATEC PSP Spin Stool DNA Plus Kit, the second extraction method used in this study."; main text also names "the STRATEC PSP Spin Stool DNA Plus Kit" - CONFIRMED | every tube was sequenced for a stretch of the bacterial 16S rRNA gene | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "For each sample, the 16S rRNA gene was amplified using Golay-barcoded universal primers 27F and 338R"; "bar-coded primers flanking the V1V2 region of the 16S rRNA gene" - CONFIRMED | a tube that held only the kit's reagents | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "Thirdly, samples containing the purification reagents only were purified by each method to document bacterial sequences introduced during downstream sample processing ("extraction blank")." - CONFIRMED | unopened sterile swab 4 | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML and https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=biosample (the 69 SAMN ids of PRJNA309332) with https://www.ebi.ac.uk/ena/portal/api/filereport?accession=PRJNA309332&result=read_run | Table 1: "Sterile swab / Unopened sterile swab"; live BioSample SAMN04431559: alias unopened.swab.4, SubjectID 4, isolation_source Sterile Swab, kit Mobio Powersoil, run SRR3110087 - CONFIRMED | The reads are in NCBI's Sequence Read Archive as project PRJNA309332 | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "The data set supporting the results of this article is available in the NCBI SRA repository, PRJNA309332" - CONFIRMED | its sample records list 28 MO BIO placental and control samples: 6 extraction blanks, 5 unopened sterile swabs, 5 air swabs, 6 fetal-side and 6 maternal-side biopsies | https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=biosample (the 69 SAMN ids of PRJNA309332) with https://www.ebi.ac.uk/ena/portal/api/filereport?accession=PRJNA309332&result=read_run | recounted from the live records 2026-11-02: Mobio Powersoil = Extraction Blank 6, Sterile Swab 5, OR Air Swab 5, Placenta (FS) 6, Placenta (MS) 6, saliva 6; lauder.json runs agree on kit, type, subject and reads for all 69 - CONFIRMED | The paper's own Figure 2b draws the same 28 columns, and in it sterile swab 4 is the one column that is red in the row labelled Cyanobacteria o__Streptophyta. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/supplementaryFiles | Figure 2 from the live zip (40168_2016_172_Fig2_HTML.jpg), viewed enlarged: panel b columns Extraction Blank 1-6, Sterile Swab 1-5, Air Swab 1-5, FS 55-70, MS 55-70 (28), then Saliva 6; in the row "Cyanobacteria o__Streptophyta" the only red cell is Sterile Swab 4 - CONFIRMED | Its run read counts average 25,044.6 per MO BIO run (851,516 over 34 runs) and 59,197.9 per PSP run (2,071,925 over 35 runs) | https://www.ebi.ac.uk/ena/portal/api/filereport?accession=PRJNA309332&result=read_run | live read_count joined to live BioSample samp_mat_process: Mobio Powersoil 851,516 over 34 runs = 25,044.59; PSP 2,071,925 over 35 = 59,197.86 (the paper prints 25,044, the truncated mean; the page shows both as they are) - CONFIRMED | and the paper prints an average of 59,198 (PSP extraction) and 25,044 (MO BIO extraction) 16S rRNA gene reads | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim: "Each sample returned an average of 59,198 (PSP extraction) and 25,044 (MO BIO extraction) 16S rRNA gene reads." - CONFIRMED | This OTU was predominant in 22 out of 23 samples, including fetal side and maternal side placenta, extraction blanks, unopened sterile swabs, and swabs exposed to air in the clinical laboratory. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim character for character, including the no-break space in "100 %" (Results, paragraph on the MO BIO kit) - CONFIRMED | The paper names this kit's background: For samples purified using the PSP kit, all placental samples and background controls contained Sediminibacterium, Bradyrhizobiaceae, Methylobacterium, and Propionibacterium. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim (the genus names are italic in the source) - CONFIRMED | fits the last sentence of the same paragraph: Additionally, one sample was high in a lineage of the order Streptophyta, possibly a sequence derived from chloroplasts in pollen. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim; it is the last sentence of the paragraph that opens "For DNA purified from the MO BIO kit", three sentences after "22 out of 23 samples" - OBSERVED | In 27 of the 28 tubes the largest layer is the same cluster, and in 26 it holds more than half of all the reads: 63.2% to 77.9% in every placental biopsy, and 65.8% to 71.1% in every extraction blank. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | largest in 27 of 28, over half in 26; placentas 63.2% to 77.9%, extraction blanks 65.8% to 71.1% - OBSERVED | made up 63% to 78% of the reads from every placental biopsy extracted with one kit, and 66% to 71% of the reads from every extraction blank | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | 63.2/77.9/65.8/71.1 round to 63/78/66/71 - OBSERVED | reads within 3% of one DNA sequence, a sequence identical to a stretch of C. difficile 16S | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | lauder.json method "Levenshtein edits <= floor(0.03 x longer length)", a read joins the first cluster whose FOUNDER is within reach, so cluster 0 is exactly the reads within 3% of the 297 bases; --deep recount of the six blanks from ENA matched (233 passed); the 297 bases sit exactly in NR_113132.1 (live NCBI, recomputed) - OBSERVED | The one tube where it is not the largest is unopened sterile swab 4, where it holds 6.5% and another cluster holds 40.6%. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | SRR3110087 (unopened.swab.4): cluster 0 at 6.5%, another cluster at 40.6% - OBSERVED | The other tube where it is under half is air swab 5, at 33.9%. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | SRR3110063 (air.swab.5): cluster 0 at 33.9%, the only other tube under half - OBSERVED | none of the 358,808 reads from the 23 placental and control tubes extracted with that kit falls in it | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | the 23 PSP placental and control runs hold 358,808 reads, 0 of them in cluster 0 - OBSERVED | A different pair of clusters leads instead: one of the two is the largest layer in all 11 PSP controls and in 10 of the 12 PSP placental tubes. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | two clusters lead all 11 PSP controls and 10 of the 12 PSP placental runs - OBSERVED | The other 2, both maternal-side biopsies, are each led by another cluster, and both come back below. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | the other 2 PSP placental runs are maternal side; the verifier ties them to the 20.6%/26.4% residues and the 66.0% cluster - CONFIRMED | A different pair of clusters leads instead | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | Abstract: "Two different DNA purification methods were compared with similar conclusions, though the composition of the contamination background differed."; Results: "results diverged radically and were associated with the kit used to purify the DNA" (the pair of clusters itself is the recount, psp-rack) - OBSERVED | With the study's other kit none of 358,808 reads came that close. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | 0 of the 358,808 PSP reads in cluster 0 - OBSERVED | Recounted from those reads, the cluster is the largest in 27 of the 28 tubes and more than half of the reads in 26. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | 27 and 26 of 28 - OBSERVED | The cluster is founded by its most frequent read, a sequence 297 bases long. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | rebuild log: "Lauder: wrote lauder.json (contaminant cluster 0, 297 bases)"; cluster numbers follow founding order by pooled abundance - OBSERVED | Reads identical to it are 25.3% to 32.3% of the reads from each MO BIO placental biopsy and 25.3% to 28.6% of those from each extraction blank; the rest of the cluster is near-copies, within 3% of it. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | reads identical to the 297 bases: placentas 25.3% to 32.3%, blanks 25.3% to 28.6%, never more than the cluster - CONFIRMED | All 297 bases match RefSeq record NR_113132.1, part of the 16S ribosomal RNA gene of Clostridioides difficile strain ATCC 9689 | https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=631251934,631250975&rettype=fasta | live 2026-11-02: ">NR_113132.1 Clostridioides difficile ATCC 9689 = DSM 1296 strain JCM 1296 16S ribosomal RNA, partial sequence" - CONFIRMED | starting at its base 21, and record NR_112172.1 from the same strain, starting at its base 13 | https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&rettype=fasta (the 47 ids of the live esearch) | my own search in the live records: the 297 bases occur exactly from base 21 of NR_113132.1 and from base 13 of NR_112172.1 ("Clostridioides difficile ATCC 9689 = DSM 1296 16S ribosomal RNA, partial sequence"); refs.json equals the live sequences - CONFIRMED | Of the 47 RefSeq 16S records that NCBI files under the family Peptostreptococcaceae | https://eutils.ncbi.nlm.nih.gov/entrez/eutils/esearch.fcgi?db=nuccore&term=Peptostreptococcaceae%5BOrganism%5D+AND+33175%5BBioProject%5D&retmax=1000&retmode=json | live 2026-11-02: "count":"47"; the 47 live sequences equal research/the-microbiome-in-the-blank/refs/refseq-peptostreptococcaceae-16s.fasta - CONFIRMED | none but the two of C. difficile comes closer than 21 edits in 297 (92.9% identity); the nearest is Romboutsia ilealis, NR_125597.1. | https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&rettype=fasta (the 47 ids of the live esearch) | my own semi-global edit distance (Python, written apart from the verifier) against the 47 live records: 0 and 0 (the two C. difficile), 21 NR_125597.1 Romboutsia ilealis, then 22 (three records); 1 - 21/297 = 92.93% - CONFIRMED | Romboutsia ilealis, NR_125597.1 | https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=nuccore&id=NR_125597.1&rettype=fasta&retmode=text | live header: "NR_125597.1 Romboutsia ilealis strain CRIB 16S ribosomal RNA, partial sequence" - CONFIRMED | However, as C. difficile only appeared in negative controls worked up with the MO BIO kit, we tentatively associate this contamination with the kit. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim (C. difficile italic in the source) - OBSERVED | a sequence identical to C. difficile 16S appeared only in samples extracted with that kit | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | cluster 0 in all 34 MO BIO runs and 0 reads in all 29 recounted PSP runs; the paper: "as C. difficile only appeared in negative controls worked up with the MO BIO kit" - OBSERVED | In this deposit it is in all 28 MO BIO placental and control tubes, and in all 6 MO BIO saliva samples at 3 to 268 reads; in the 29 PSP runs recounted here it has no reads at all. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | cluster 0 in all 34 MO BIO runs, the 6 MO BIO salivas at 3 to 268 reads, 0 in all 29 PSP runs - OBSERVED | Three more clusters hold shorter copies of the same stretch, 255 to 282 bases long; the 97% rule keeps them apart because their lengths differ by more than 3%. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | lauder.json contaminant.shorter: clusters 86, 90, 813 of 282, 255, 263 bases, each a prefix of the 297 at offset 20; 297 - 282 = 15 edits > floor(0.03 x 297) = 8 - OBSERVED | Adding them in changes neither the 27 nor the 26, and none of them occurs in a PSP run. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | with clusters 86, 90 and 813 added, still 27 and 26, and none in a PSP run - OBSERVED | the MO BIO placentas keep 1.2% to 6.0% of their reads and its controls keep 1.6% to 42.1%: extraction blanks 1.6% to 5.8%, air swabs 1.7% to 14.1%, sterile swabs 1.9% to 42.1%. | recreated: Playwright render of the shipped files (requests route-intercepted, no server) plus the rebuild above | status at the defaults: "MO BIO: placentas keep 1.2% to 6.0% of their reads; controls keep 1.6% to 42.1% (extraction blanks 1.6% to 5.8%)"; the verifier recomputes the air and sterile ranges - OBSERVED | Its controls keep 1.3% to 8.2%, but its placentas keep 3.0% to 26.4%, and three maternal-side biopsies keep 14.2%, 20.6% and 26.4%. | recreated: Playwright render of the shipped files (requests route-intercepted, no server) plus the rebuild above | status: "PSP: placentas keep 3.0% to 26.4% of their reads; controls keep 1.3% to 8.2%"; the verifier asserts 14.2%, 20.6% and 26.4% - CONFIRMED | A read whose sequence occurs only once in the whole study and joins no cluster counts in its tube's total but is never counted as left. | public/strata/the-microbiome-in-the-blank/engine.mjs | residue() adds only cluster counts and divides by x.reads, which includes unclustered reads; build.mjs: "A sequence read only once that matches no cluster stays unclustered (it still counts in its run's total)" - CONFIRMED | The authors saw the same thing: apparently enriched lineages could be found readily in comparisons between contamination data sets | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim; context: "It is unlikely that we fully sampled all potential contaminants, and apparently enriched lineages could be found readily in comparisons between contamination data sets." - CONFIRMED | The paper also reports maternal-side samples high in lineages of their own, looked for those in the same women's saliva and vaginal samples | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "One of the maternal side biopsy samples was high in Enterobacteraceae and a second in Prevotella."; "the OTU enriched in the maternal side placental biopsy was not among those present in the subject's saliva sample, nor in her vaginal or fetal side placental samples"; "no reads were detected from this OTU in saliva or vaginal samples" - CONFIRMED | and wrote: Thus, we were unable to trace the origin of these outlier lineages to specific body sites in our paired samples. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim - CONFIRMED | The authors' own test starts from a sound idea: We reasoned that authentic microbiota should be mostly reproducible regardless of the DNA extraction kit used | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim (Discussion; "Fig. 4" has a no-break space in the source) - CONFIRMED | They ran the same test on saliva, where bacteria certainly live | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "In contrast, the same filtering procedure recovered 28 OTUs from salivary samples."; Discussion: "Oral sites are known to host dense bacterial communities" - CONFIRMED | for saliva, each kit took a portion of the same sample | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | Method 1: "One-hundred microliters of each saliva sample was used for DNA extraction." - CONFIRMED | for saliva, each kit took a portion of the same sample | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | Method 2: "Seventy to 100 μL of each saliva sample was used for DNA extraction." - CONFIRMED | for saliva, each kit took a portion of the same sample | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "Each participant was asked to allow saliva to collect in her mouth for 1 min, after which she spit into the 50-mL tube." - CONFIRMED | while for placenta each took a different biopsy | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | Method 2: "DNA was isolated from replicate biopsies, saliva, and control samples using the MO BIO PowerSoil DNA Isolation Kit" - CONFIRMED | and among its limits the paper writes: If there is patchy microbial colonization, it might have gone undetected. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim, in the paragraph that opens "This study has several limitations." - OBSERVED | At the paper's threshold of 1%, saliva passes 25 clusters and placenta none. | recreated: Playwright render of the shipped files (requests route-intercepted, no server) plus the rebuild above | status at load: "At T = 1%: saliva passes 25 clusters; placenta passes 0 (fetal side 0, maternal side 0)." - CONFIRMED | The paper reports the same shape: There were no placental OTUs that achieved at least 1 % abundance in both PSP and MOBIO in at least one sample and not in controls | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim, including the source's "controls, In contrast" and the no-break space in "1 %" - OBSERVED | At 0.5% saliva passes 31 and placenta 1; at 2%, 19 and 0. Lower the threshold and placental clusters appear: 2 at 0.1%, 11 at 0.01%, 18 at 0.001%. | recreated: Playwright render of the shipped files (requests route-intercepted, no server) plus the rebuild above | statuses: 0.1% "placenta passes 2", 0.01% "placenta passes 11", 0.001% "placenta passes 18", 2% "saliva passes 19 clusters; placenta passes 0"; 0.5% (31 and 1) asserted by the verifier - OBSERVED | 29 of the 31 bars end below 0.2%. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | 31 placental windows, 29 with an upper end below 0.2% - OBSERVED | The closest call at 1% is one cluster in one woman's maternal-side biopsy: 3.7% of the PSP reads and 0.62% of the MO BIO reads, while no control holds more than 0.37% of it. | recreated: Playwright render of the shipped files (requests route-intercepted, no server) plus the rebuild above | status: "maternal side, 3.72% of PSP reads and 0.616% of MO BIO reads, highest control 0.373%; it passes for T above 0.373% up to 0.616%"; 3.72/0.616 = 6.0 - OBSERVED | That cluster holds 66.0% of the PSP reads from one woman's maternal-side biopsy and 3.1% of the MO BIO reads from a replicate biopsy of the same side, so it clears 1% in both kits. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | cluster 1: 66.0% of the PSP maternal-side run, 3.1% of the MO BIO replicate, 3.6% in a MO BIO extraction blank (the next sentence, "It fails because a MO BIO extraction blank holds 3.6% of it", is in this entry's excerpt) - CONFIRMED | Its Results say 16 in the maternal side placental samples, and 5 in the fetal side placental samples. Two OTUs appeared in both fetal and maternal side samples, leaving 19 unique placental OTUs. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim; the sentence opens "Analyzing shared OTUs achieving 0.001 % in any sample, we found that 2197 OTUs were recovered in the oral samples," - CONFIRMED | Its Discussion says we found 18 OTUs reproducible between extractions for the maternal side sample and 5 for the fetal side sample | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim, opening "At the lowest cut value, 0.001 % abundance," - CONFIRMED | and its Table S3 lists 21 clusters, 18 maternal-side and 5 fetal-side with 2 in both. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/supplementaryFiles | UNREADABLE to the tool (zip); read from the live zip 2026-11-02, 40168_2016_172_MOESM5_ESM.doc as text: 21 denovo rows; Placenta (MS) nonzero in 18, Placenta (FS) in 5, both in 2 (denovo15726, denovo7082); identical to the kept copy - OBSERVED | This recount finds 18 at 0.001%: 15 maternal-side and 5 fetal-side, 2 in both. | recreated: Playwright render of the shipped files (requests route-intercepted, no server) plus the rebuild above | status at 0.001%: "placenta passes 18 (fetal side 5, maternal side 15)"; 15 + 5 - 18 = 2 in both - CONFIRMED | The authors read their low-level clusters with care: Some of these extremely low-abundance lineages may represent sporadic, rare placental microbiota. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim; the em dash in "data—several" is the source's - CONFIRMED | In 2018 Nicole Davis and colleagues, who wrote the decontam software, ran it on this dataset. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC6298009/fullTextXML | "Here we introduce decontam (https://github.com/benjjneb/decontam), an open-source R package"; "We used the prevalence method as implemented in the isNotContaminant function to further explore the possibility that some ASVs in the Lauder et al. dataset could be consistent with placental origin"; Crossref: first author Nicole M. Davis, issued 2018-12 - CONFIRMED | Its prevalence method rests on the observation that sequences from contaminating taxa are likely to have higher prevalence in control samples than in true samples | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC6298009/fullTextXML | verbatim: "(2) sequences from contaminating taxa are likely to have higher prevalence in control samples than in true samples", one of "two simple de novo classification methods based on widely reproduced signatures of external contamination" - CONFIRMED | and they used amplicon sequence variants (ASVs), inferred by DADA2, rather than 97% clusters | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC6298009/fullTextXML | "Demultiplexed fastq files from the oral, Salter et al.'s, and placenta datasets were then processed into amplicon sequence variants (ASVs) by DADA2 version 1.8.0" - CONFIRMED | Five of those six ASVs matched Homo sapiens rather than any bacterial taxa. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC6298009/fullTextXML | verbatim ("Table 1" has a no-break space and Homo sapiens is italic in the source) - CONFIRMED | The sixth was a Ruminococcaceae variant, a known member of human gut microbial communities | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC6298009/fullTextXML | "The other putative non-contaminant ASV was a Ruminococcaceae variant, a known member of human gut microbial communities [2], but we are unable to establish its ground truth." (both fragments the page quotes come from this sentence) - OBSERVED | One cluster in this recount does set the placentas apart: it is in 18 of the 24 placental extractions and in none of the 27 controls, at most 0.64% of a tube's reads. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | cluster 454: in 18 of 24 placental extractions, 0 of 27 controls, at most 0.64% - CONFIRMED | Of its own clusters found in more than one placenta, the Lauder paper wrote: The three OTUs found in multiple placentas could not be assigned to any bacterial taxa, and further analysis showed the sequences were 100 % identical to human chromosomal sequences. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim; preceded by "Of these OTUs, 16 OTUs were seen in one placenta only and three were seen in multiple placentas." - CONFIRMED | In 2014 Susannah Salter and colleagues made the mechanism visible. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | author list opens "Salter Susannah J"; Crossref 10.1186/s12915-014-0087-z: BMC Biology 12, article 87, issued 2014-11-12 - CONFIRMED | They grew a pure culture of Salmonella bongori | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | "we used 16S rRNA gene sequence profiling of a pure culture of Salmonella bongori that had undergone five rounds of serial ten-fold dilutions" - CONFIRMED | a species picked because we have not observed it as a contaminant in any of our previous studies | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | verbatim: "S. bongori was chosen because we have not observed it as a contaminant in any of our previous studies" - CONFIRMED | had Imperial College London, the University of Birmingham and the Wellcome Trust Sanger Institute each extract every dilution and amplify it, with different production batches of one kind of kit | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | "each of the three institutes (Imperial College London, ICL; University of Birmingham, UB; Wellcome Trust Sanger Institute, WTSI) extracted DNA from the S. bongori aliquots in parallel, using different production batches of the FastDNA Spin Kit For Soil"; "Each aliquot was extracted once at each institute."; "The PCR amplification was carried out ... at WTSI, ICL and UB" - CONFIRMED | They estimate about 10^8 cells went into the undiluted extraction and about 10^3 into the fifth dilution. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | "equating to a range of approximately 10^8 cells as input for DNA extraction in the original undiluted sample, to 10^3 cells in dilution five" (superscripts in the XML) - OBSERVED | the share classified as S. bongori falls from 95.3%, 95.2% and 94.6% in the three undiluted cultures to 3.9%, 19.8% and 6.3% at the fifth dilution (Imperial, Birmingham, Sanger). | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | salter.json: dilution 0 shares 95.33%, 95.22%, 94.61%; dilution 5 shares 3.90%, 19.77%, 6.34%; Table S1a (live): NF1/1/SAL-1 are dilution 0, NF6/6/SAL-6 dilution 5, COX = ICL, LOM = UB, SAL = WTSI - OBSERVED | The undiluted cultures fall short of 100% mostly because of the 8-edit limit: in the three of them, 4.0%, 4.0% and 4.8% of the reads have first 150 bases more than 8 edits from the S. bongori record. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | reads beyond 8 edits in the undiluted runs: 4.01%, 4.03%, 4.79% - OBSERVED | The rule also asks that a read be nearer to that record than to any of 122 other Enterobacteriaceae records, which sets aside another 0.6% to 0.7%. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | salter.json method: panel 253, usable 123, rivals 122; the nearer-record clause sets aside 0.65%, 0.74%, 0.61% - OBSERVED | at the Sanger Institute the second dilution sits below the third, 45.3% against 63.1%. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | SAL_3_40 (dilution 2) 45.28%, SAL_4_40 (dilution 3) 63.11% - CONFIRMED | Salter's own summary: S. bongori was the sole organism identified in the original undiluted culture | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | verbatim ("Figure 1" has a no-break space in the source) - CONFIRMED | The curve is the simplest mixture, as the decontam paper sets it out: Let total sample DNA (T) be a mixture of two components (T = C + S) | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC6298009/fullTextXML | verbatim in its words; the source sets "T = C + S" with thin spaces and italic letters, the page with plain spaces - OBSERVED | The sample's share of the reads is then S/(S + C), half when S equals C. The points follow it only roughly; no single C fits all three laboratories, or every dilution of one. | recreated: python over salter.json, implied C = S(1 - f)/f with S = 10^(8 - dilution) | implied C spans 293-fold (ICL), 1236-fold (UB) and 386-fold (WTSI) within each laboratory; S/(S + C) = 1/2 at S = C is arithmetic - CONFIRMED | By quantitative PCR, the Lauder study found that placental and control samples showed low and indistinguishable numbers of 16S rRNA gene copies | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "For both purification methods, we conclude that placental and control samples showed low and indistinguishable numbers of 16S rRNA gene copies." - CONFIRMED | the 2014 study tested DNA extraction kits from four different manufacturers | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | "DNA extraction kits from four different manufacturers were used in order to investigate whether or not the problem was limited to a single manufacturer." (the shotgun-metagenomics experiment, kits FP, MB, QIA and PSP, processed at UB) - CONFIRMED | and found that Regardless of kit, contamination was always the predominant feature of the sequence data by the fourth serial dilution | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | verbatim; continues "which equated to an input of around 10^4 Salmonella cells" - CONFIRMED | In 2014 Aagaard and colleagues, working from placental specimens of 320 subjects | https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:24848255%20AND%20SRC:MED&format=json&resultType=core | "A population-based cohort of placental specimens collected under sterile conditions from 320 subjects with extensive clinical data was established"; pubYear 2014 - CONFIRMED | We characterized a unique placental microbiome niche, composed of nonpathogenic commensal microbiota from the Firmicutes, Tenericutes, Proteobacteria, Bacteroidetes, and Fusobacteria phyla. | https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:24848255%20AND%20SRC:MED&format=json&resultType=core | verbatim from the abstract - CONFIRMED | The Lauder paper tested that idea, and its own limits paragraph says: Our data do not rule out the existence of placental microbiota | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | verbatim; the sentence after "This study has several limitations."; the em dash is the source's - CONFIRMED | The Lauder paper tested that idea | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "In the report of a commensal placental microbiome [3], only a partial set of control studies was reported, leaving the possible contribution of contamination unclear."; ref 3 is Aagaard et al., The placenta harbors a unique microbiome, Sci Transl Med 2014; MO BIO is the kit "used by Aagaard et al. [3]" - CONFIRMED | In 2019 de Goffau and colleagues concluded | https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:31367035%20AND%20SRC:MED&format=json&resultType=core | title "Human placenta has no microbiome but can contain potential pathogens."; Crossref: Nature 572, 329-334, issued 2019-07-31 - MINOR | concluded that the human placenta does not have a microbiome | https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:31367035%20AND%20SRC:MED&format=json&resultType=core | the words are verbatim, but the page's "with one exception:" makes the exception qualify the no-microbiome conclusion; in the abstract it qualifies "Almost all signals were related either to the acquisition of bacteria during labour and delivery, or to contamination of laboratory reagents with bacterial DNA.", and the conclusion reads "the human placenta does not have a microbiome, but it does represent a potential site of perinatal acquisition of S. agalactiae" - CONFIRMED | with one exception: The exception was Streptococcus agalactiae (group B Streptococcus), for which non-contaminant signals were detected in approximately 5% of samples collected before the onset of labour. | https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:31367035%20AND%20SRC:MED&format=json&resultType=core | verbatim; the sentence follows "Almost all signals were related either to the acquisition of bacteria during labour and delivery, or to contamination of laboratory reagents with bacterial DNA." - CONFIRMED | In 2023 a review by Kennedy and colleagues began | https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:36697862%20AND%20SRC:MED&format=json&resultType=core | "Here we evaluate recent studies that characterized microbial populations in human fetuses"; Europe PMC pubType includes "Review"; Crossref: Nature 613, 639-649, issued 2023-01-25 - CONFIRMED | Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate. | https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:36697862%20AND%20SRC:MED&format=json&resultType=core | verbatim; the first sentence of the abstract - CONFIRMED | Its own reading: Our analysis indicates that the detected microbial signals are likely the result of contamination | https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:36697862%20AND%20SRC:MED&format=json&resultType=core | verbatim - OBSERVED | In this study, the cluster that fills a MO BIO tube that held nothing fills every MO BIO placental tube too, and the test that saliva passes at 1%, placenta does not. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | cluster 0 over half of all 12 MO BIO placental runs and all 6 extraction blanks; agreement test at 1%: saliva 25, placenta 0 - CONFIRMED | The paper clustered with UCLUST in QIIME and named clusters from GreenGenes | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "Operational taxonomic units (OTUs) were created by clustering the reads at 97 % identity using UCLUST"; "analyzed using the QIIME software package"; "Taxonomic assignments were generated by the UCLUST consensus method of QIIME 1.8, using the GreenGenes 16S rRNA gene database v. 13_8" - OBSERVED | The deposited reads are already merged pairs, and their counts give the printed means, so they are not filtered again. | recreated: python over the 63 md5-checked Lauder FASTQs (one file per run in the live ENA report) | every read 234 to 461 bases (longer than a 250-base MiSeq read, so merged); run counts give 25,044.6 and 59,197.9 against the printed 25,044 and 59,198 - OBSERVED | by walking the 421,286 distinct read sequences from most to least frequent | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | rebuild log: "Lauder: 63 runs, 2127022 reads, 421286 unique sequences" - CONFIRMED | For Salter's reads (the 21 runs at 40 PCR cycles, forward reads only) | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/supplementaryFiles | UNREADABLE to the tool (zip); read from the live zip 2026-11-02, 12915_2014_87_MOESM1_ESM.docx: Table S1a "Summary of 16S rRNA gene sequence samples from project accession ERP006737." lists 21 runs at 40 PCR cycles (COX-NF1-40 to COX-NF-NEG-40, LOM-1-40 to LOM-PCR-40, SAL-1-40 to SAL-PCR-40); the kept copy differs only in quote marks - CONFIRMED | within 8 edits of record NR_074888.1, the strain cultured | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | "Salmonella bongori strain NCTC-12419 was cultured overnight on Luria-Bertani (LB) plates"; live NCBI header "NR_074888.1 Salmonella bongori strain NCTC 12419 16S ribosomal RNA, complete sequence" - CONFIRMED | every other usable record in NCBI's RefSeq 16S collection for the Enterobacteriaceae | https://www.ncbi.nlm.nih.gov/bioproject/33175 | live: "The RefSeq dataset contains curated 16S ribosomal RNA sequences that correspond to bacteria type materials." - OBSERVED | which match Citrobacter koseri records exactly over their first 200 bases. | recreated: python over ERR584348_1.fastq.gz (SAL_PCR_40, md5-equal to ENA) against the 253 live Enterobacteriaceae records | the two most frequent first-200-base sequences (3,037 and 1,899 of 39,940 reads) occur exactly in NR_118105.1 and NR_104890.1, both Citrobacter koseri - CONFIRMED | A. P. Lauder, A. M. Roche, S. Sherrill-Mix and others, Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota, Microbiome 4 (2016) 29 | https://api.crossref.org/works/10.1186/s40168-016-0172-3 | Abigail P. Lauder; Aoife M. Roche; Scott Sherrill-Mix (+7); title as printed; Microbiome vol 4, article 29, 2016 - CONFIRMED | Creative Commons Attribution 4.0; its data CC0 | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | "distributed under the terms of the Creative Commons Attribution 4.0 International License"; "The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated." - CONFIRMED | NCBI itself places no restrictions on their use. | https://www.ncbi.nlm.nih.gov/home/about/policies/ | live: "NCBI itself places no restrictions on the use or distribution of the data contained therein." - OBSERVED | of which the 63 used here (all but the six vaginal swabs, which went through one kit only) were fetched from the European Nucleotide Archive with each file's md5 checked | research/the-microbiome-in-the-blank/fetch.mjs; recreated: md5 of the 84 scout FASTQs against live ENA fastq_md5 | 84 of 84 match; fetch.mjs drops isolation_source Vaginal Swab and checks md5; the paper: "Vaginal samples were collected as single swabs and thus could be assayed with only one DNA purification method."; 69 - 6 = 63 - CONFIRMED | the 253 found by Enterobacteriaceae[Organism] AND 33175[BioProject] | https://eutils.ncbi.nlm.nih.gov/entrez/eutils/esearch.fcgi?db=nuccore&term=Enterobacteriaceae%5BOrganism%5D+AND+33175%5BBioProject%5D&retmax=1000&retmode=json | live: "count":"253"; the 253 live sequences equal research/the-microbiome-in-the-blank/refs/refseq-enterobacteriaceae-16s.fasta - CONFIRMED | S. J. Salter, M. J. Cox, E. M. Turek and others, Reagent and laboratory contamination can critically impact sequence-based microbiome analyses, BMC Biology 12 (2014) 87 | https://api.crossref.org/works/10.1186/s12915-014-0087-z | Susannah J Salter; Michael J Cox; Elena M Turek (+7); title as printed; BMC Biology vol 12, article 87, 2014 - CONFIRMED | reads at ENA ERP006737. Creative Commons Attribution 4.0. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | "This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)" - CONFIRMED | reads at ENA ERP006737 | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4228153/fullTextXML | "Data are deposited under ENA project accession EMBL: ERP006737" - CONFIRMED | N. M. Davis, D. M. Proctor, S. P. Holmes, D. A. Relman and B. J. Callahan, Simple statistical identification and removal of contaminant sequences in marker-gene and metagenomics data, Microbiome 6 (2018) 226 | https://api.crossref.org/works/10.1186/s40168-018-0605-2 | Nicole M. Davis; Diana M. Proctor; Susan P. Holmes; David A. Relman; Benjamin J. Callahan (5 authors); title as printed; Microbiome vol 6, article 226, 2018 - CONFIRMED | Microbiome 6 (2018) 226. Creative Commons Attribution 4.0. | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC6298009/fullTextXML | "distributed under the terms of the Creative Commons Attribution 4.0 International License" - CONFIRMED | K. Aagaard, J. Ma, K. M. Antony and others, The placenta harbors a unique microbiome, Science Translational Medicine 6 (2014) 237ra65 | https://api.crossref.org/works/10.1126/scitranslmed.3008599 | Kjersti Aagaard; Jun Ma; Kathleen M. Antony (+3); "The Placenta Harbors a Unique Microbiome"; Science Translational Medicine vol 6, 2014; Europe PMC pageInfo 237ra65 - CONFIRMED | M. C. de Goffau, S. Lager, U. Sovio and others, Human placenta has no microbiome but can contain potential pathogens, Nature 572 (2019) 329-334 | https://api.crossref.org/works/10.1038/s41586-019-1451-5 | Marcus C. de Goffau; Susanne Lager; Ulla Sovio (+6); title as printed; Nature 572, 329-334, 2019 - CONFIRMED | K. M. Kennedy, M. C. de Goffau, M. E. Perez-Muñoz and others, Questioning the fetal microbiome illustrates pitfalls of low-biomass microbial studies, Nature 613 (2023) 639-649 | https://api.crossref.org/works/10.1038/s41586-022-05546-8 | Katherine M. Kennedy; Marcus C. de Goffau; Maria Elisa Perez-Muñoz (+43); title as printed; Nature 613, 639-649, 2023 - CONFIRMED | A placental biopsy sits at the low end of that axis. (not in the source record) | https://www.ebi.ac.uk/europepmc/webservices/rest/PMC4917942/fullTextXML | Results: "In summary, all placental and negative control samples were at the extreme low end of the range detectable by qPCR"; the page's next sentence quotes the qPCR conclusion (q-qpcr) - OBSERVED | Text app.mjs builds at load and on use: tube labels, legend, the four status lines, the sequence box (not in the source record) | recreated: Playwright render of the shipped files, requests route-intercepted (no server), then Reveal, PSP, subtraction PSP, sequence toggle and five thresholds | "The amber cluster is the largest layer in 27 of the 28 and over half the reads in 26."; "PSP kit: 12 of the 23 tubes are placental biopsies and 11 are controls ... The amber cluster has 0 of the 358,808 reads in these tubes."; "Recounted at the fifth dilution: Imperial 3.9%, Birmingham 19.8%, Sanger 6.3%."; "297 of 297 bases identical: yes"; every figure agrees with the prose; no external request, no console error - CONFIRMED | plain: whether a healthy placenta has its own bacteria is still debated | https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:36697862%20AND%20SRC:MED&format=json&resultType=core | "Whether the human fetus and the prenatal intrauterine environment (amniotic fluid and placenta) are stably colonized by microbial communities in a healthy pregnancy remains a subject of debate." - OBSERVED | plain: With one DNA kit, near-copies of one scrap of bacterial DNA made up most of the results from placentas and empty tubes alike; with the other kit they were missing. | recreated: node research/the-microbiome-in-the-blank/build.mjs --lauder <63 scout FASTQs> --salter <21 scout FASTQs> --out (all 84 files md5-equal to ENA live fastq_md5; 6 min 30 s), then cmp: lauder.json, salter.json and refs.json byte-identical to the shipped files; AW_DIR= node verify-the-microbiome-in-the-blank.mjs asserts the figure from them (221 passed, 0 failed), and my own Python over lauder.json gives it again | extraction blanks 65.8% to 71.1%, placentas 63.2% to 77.9% with MO BIO; 0 of 358,808 PSP reads - OBSERVED | With --deep it also downloads the six MO BIO extraction blanks from the European Nucleotide Archive and recounts them from the raw reads: each run's total, and the reads within 3% of the 297-base sequence. | recreated: AW_DIR= node verify-the-microbiome-in-the-blank.mjs --deep, run twice from a directory holding only the verifier | first run: ENA answered 503 for SRR3110032 and SRR3110035 (exit 1, 229 passed, 2 failed); second run: exit 0, 233 passed, 0 failed - CONFIRMED | relates notes: the-ten-to-one-person (how many bacteria a body carries), mites-that-live-on-your-face (the method decides how many people have face mites), smaller-than-we-could-see (a null result and its positive controls) | src/content/strata/the-ten-to-one-person.md, src/content/strata/mites-that-live-on-your-face.md, src/content/strata/smaller-than-we-could-see.md | all three exist; their deks: "38.64 trillion bacteria for a specified ... reference man"; "counting mites under a microscope found them on just 14% of adults; screening ... for Demodex DNA found them on 100%"; "Every positive control here is planted ..." ## Findings - [fixed] MINOR: the de Goffau sentence made the paper's exception qualify its no-microbiome conclusion. The page read (index.html:202): "In 2019 de Goffau and colleagues concluded that the human placenta does not have a microbiome, with one exception: The exception was Streptococcus agalactiae (group B Streptococcus), for which non-contaminant signals were detected in approximately 5% of samples collected before the onset of labour." Re-read in the kept copy (research/the-microbiome-in-the-blank/sources/degoffau2019-abstract.txt) and the live Europe PMC abstract (https://www.ebi.ac.uk/europepmc/webservices/rest/search?query=EXT_ID:31367035%20AND%20SRC:MED&format=json&resultType=core, fetched 2026-11-02), which agree word for word: "Almost all signals were related either to the acquisition of bacteria during labour and delivery, or to contamination of laboratory reagents with bacterial DNA. The exception was Streptococcus agalactiae (group B Streptococcus), for which non-contaminant signals were detected in approximately 5% of samples collected before the onset of labour." The exception is to the sentence about signals; the conclusion keeps the two apart: "the human placenta does not have a microbiome, but it does represent a potential site of perinatal acquisition of S. agalactiae". The page now reads: "In 2019 de Goffau and colleagues concluded that the human placenta does not have a microbiome. Of the bacterial DNA signals they did detect, they wrote: Almost all signals were related either to the acquisition of bacteria during labour and delivery, or to contamination of laboratory reagents with bacterial DNA. The exception was Streptococcus agalactiae (group B Streptococcus), for which non-contaminant signals were detected in approximately 5% of samples collected before the onset of labour." (both sentences in one quotation, data-src degoffau-gbs). quotes.json degoffau-gbs passage and the q-gbs entry of assay/sources/the-microbiome-in-the-blank.json (claim, quote, excerpt) updated to match. After the fix: node verify-the-microbiome-in-the-blank.mjs 293 passed, 0 failed (221 from outside with AW_DIR); a scratch copy with "Almost every signal was related" fails the quotation check (219 passed, 2 failed). - [fixed] MINOR (fix-checker, after round 2): the generator and README no longer described the shipped quotes.json. Round 2 extended the degoffau-gbs passage in public/strata/the-microbiome-in-the-blank/quotes.json (md5 f940b925fa0ea1088a0d0eb2b307c6b7) but research/the-microbiome-in-the-blank/make-quotes.py still began that passage "The exception was Streptococcus agalactiae", so rerunning it wrote the round-1 file (06edf8648a7ee897c904ed9daae72030) and the verifier then failed, while README.md said 06edf8648a7ee897c904ed9daae72030 was "reproduced byte for byte". Re-read the kept copy research/the-microbiome-in-the-blank/sources/degoffau2019-abstract.txt: "Almost all signals were related either to the acquisition of bacteria during labour and delivery, or to contamination of laboratory reagents with bacterial DNA. The exception was Streptococcus agalactiae (group B Streptococcus), for which non-contaminant signals were detected in approximately 5% of samples collected before the onset of labour." make-quotes.py now carries that two-sentence passage; run on a scratch copy (python3 -I, outside the repository) it finds all 27 passages and writes f940b925fa0ea1088a0d0eb2b307c6b7, byte-identical to the shipped quotes.json. The README build log now gives that md5 "since fix round 2" with the round-1 and build md5s as history. Nothing a reader sees changed. The claim line for the old de Goffau sentence keeps the pass's original words, because the record says what the pass found.