V2 chemistry

Total RNA isolation, quantification and preparation for sequencing was as previously described [12 (link)]. RNA Integrity Numbers (RIN) averaged 6.2 and libraries (four replicate samples per treatment group) were prepared and sequenced (101 bp paired-end reads) at the University of Minnesota Genomics Center (UMGC) on the HiSeq 2000 using v2 chemistry (Illumina, Inc.). Raw data were deposited in the NCBI’s Gene Expression Omnibus (GEO) repository as SRA BioProject 346253. Analysis of RNAseq data followed the protocols outlined in Reed et al. [12 (link)].

Bacterial RNA isolation was carried out as described previously (Wehner et al., 2014 (link)). The quality of total RNA was assessed using Bioanalyzer 2100 Total RNA nano chip (Agilent, Santa Clara, CA, USA) and RNA concentration was measured using Qubit 2.0 fluorimeter (Invitrogen/Life Technologies, Carlsbad, CA, USA). rRNA was depleted from 1 μg of total RNA using Ribo-Zero Magnetic Kit (Bacteria; Epicentre, Madison, WI, USA). Depleted RNA was then treated with tobacco acid pyrophosphatase (Epicentre, Madison, WI, USA) and cleaned up with the RiboMinus concentration module (Life Technologies, Carlsbad, CA, USA).
For fragmentation and further library preparation TruSeq Stranded Total RNA Seq Kit (Illumina, San Diego, CA, USA) was used according to manufacturer’s instruction. In this process a barcoded-approach was chosen to facilitate multiplexed sequencing. For reverse transcription Superscript II polymerase (Life Technologies, Carlsbad, CA, USA) was used and libraries were purified with AMPure XP Reagent (Beckman Coulter, Pasadena, CA, USA). The yield and size distribution of the amplified cDNA were assessed with DNA High sensitivity kit (Agilent, Santa Clara, CA, USA). Libraries were then diluted to 4 nM, pooled, denatured and further diluted to 10 pM. Sequencing was carried out on the MiSeq using v2 chemistry (Illumina, San Diego, CA, USA).

First, the RNA samples were treated with RNA 5’ Polyphosphatase (Epicentre, Paris, France). Oligonucleotide adapters were ligated to the 5’ and 3’ ends of the RNA samples. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3′ adapter as a primer. The resulting cDNAs were amplified by PCR using a high-fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics, Krefeld, Germany). The yield and size distribution of the amplified cDNA were assessed with Agilent DNA High Sensitivity Kit (Agilent, Santa Clara, CA, USA). Libraries were then diluted to 4 nM, pooled, denatured, and further diluted to 10 pM. Sequencing was carried out on the MiSeq using v2 chemistry (Illumina, San Diego, CA, USA).
CLC Genomics workbench 8.5.1 (CLC bio, Qiagen, Hilden, Germany) was then utilized to align processed reads to the reference (NC_003210) and count reads. As parameters for the mapping, a mismatch cost of 2, an insertion cost of 3, a deletion cost of 3 along with a threshold for the length fraction of 0.8 and a required similarity fraction of 0.8 were chosen. The counting was carried out in a strand-specific manner. Sequences originating from this study are deposited in “JLUbox”. Link: https://jlubox.uni-giessen.de/filestable/MlJ2NnBvRmZMU0V5Y0d1RUM2V2ZL, accessed on 1 September 2022.

To generate amplicons of a low size (around 60–200 bp), 108 primer sets spanning the whole mtDNA (Supplemental Table S1) were designed according to the mt-sequence of accession no. NC_012920 or taken from previously published primer sets (Fig. 1A, Supplemental Table S1). For enrichment of the mt-genome by a multiplex PCR, primer sets were pooled in four primer mixes of 2 µM and in each reaction, 10 ng of PCR accessible DNA -representing DNA of around 1500 cells was used (Fig. 1B).
mtDNA was then amplified in four separate multiplex PCR reactions per sample using the GeneRead DNAseq Panel PCR Kit (QIAGEN Inc., Hilden, GER) in accordance with the manufacturer´s protocol. Libraries were pooled and purified using Agencourt® AMPure® XP magnetic beads and a Biomek® FXp workstation (Beckman Coulter Inc, Fullerton, CA, USA). Fifty ng enriched targets of each sample were adenylated and ligated to NEXTflex™ DNA barcodes-48 (Bioo Scientific, Austin, TX, USA). After Agencourt® AMPure® XP magnetic bead purification and size selection, barcoded libraries were amplified by five PCR cycles. Finally, 12 pM of the constructed libraries were sequenced using the V2 chemistry of Illumina Inc. (San Diego, CA, USA) and 2 × 300 bp sequencing read length on an Illumina MiSeq platform following the manufacturer’s recommendations.

Stool samples were collected from diapers using a standardized biological material collection kit (Stool Sample Application System (SAS); Immundiagnostik, Bensheim, Germany). The samples were collected by previously trained hospital staff or by the parents according to an established procedure and stored in a refrigerator (for a maximum of eight hours) before transport. Transport to the laboratory took no longer than 60 min, at 6–8 °C. The stool samples were then frozen at − 20 °C until metagenomic analyses were conducted. Microbiome DNA extraction was performed using the Genomic Mini AX Bacteria + Spin and Genomic Mini AX Soil Spin kits (A&A Biotechnology, Gdynia, Poland) following the manufacturer’s protocol. DNA concentrations were determined by fluorometry (DeNovix DS-11 FX + Spectrophotometer/Fluorometer, Wilmington, DE, USA). Samples were subsequently stored at − 20 °C. Metagenomic libraries of the V3–V4 hypervariable region of the 16S rRNA gene were constructed and further sequenced on the MiSeq platform (paired-end 2 × 250 bp) using V2 chemistry from Illumina (Illumina, San Diego, CA, USA). Next generation sequencing (NGS) was performed by Genomed S.A., Warsaw, Poland.
For validation, we used the independent Illumina MiSeq 16S rRNA data of the same hypervariable region (V3–V4) derived from the 227 stool samples of healthy term children enrolled in the HMS cohort.

The RNA extractions were carried out with the Zymo Research Soil/Fecal RNA kit (R2040, Zymo Research, Irvine, CA, USA). After lysis (bead beating), the Zymo Research kit protocol was followed. The DNA contamination was removed by Thermo Scientific Rapid Out™ DNA removal kit (K2981, Thermo Fisher Scientific, Waltham, MA, USA). Before transcriptome sequencing, rRNA was depleted from RNA by using the Gram + /Gram − depletion kit in a 60:40 ratio (RiboMinus A15020 Life Technologies, San Francisco, CA, USA). The mRNA library was prepared using the mRNA Sample Prep kit (Illumina, San Diego, CA, USA). Sequencing was performed using the Illumina V2 chemistry (2 × 250 bp) and applying the MiSeq paired-end mode. Raw sequences are available on the NCBI Sequence Read Archive (SRA) under the accession number: PRJNA922065.