16S rRNA gene amplicon sequencing of stool from an extremely premature neonate [JMF-1906-2-0325A]
Sixty extremely premature infants were enrolled between September 2017 and June 2019 at the General Hospital of Vienna as part of the PreMiBraIn study, which was approved by the ethics committee of the Medical University of Vienna (Ethics number 1348/2017). Inclusion criteria were birth before the 28th week of gestation with less than 1000 g birth weight. Infants with congenital malformations, chromosomal aberrations, maternally transmitted infectious diseases (e.g. HIV, hepatitis A/B/C, etc.) and inborn errors of metabolism were excluded. 547 stool samples from all 60 extremely premature infants were collected from diapers via collection tubes during patient care routines. Samples for extraction of nucleic acids were immediately stored at -80C until further analysis. Subsequently, samples were weighed in (20-30mg) for phenol-chloroform extraction of total nucleic acids (TNA). TNA were eluted in 40 ul nuclease free water and stored at -20C until further analysis. Extracted TNA concentration was measured using Quant-iT PicoGreen dsDNA Assay. Extracts served as templates for qPCR amplification of bacterial 16S rRNA genes, as well as for 16S rRNA gene amplicon sequencing. For the enumeration of gene copy numbers the primer pair 515F (Parada, Needham and Fuhrman, 2016) and 806R (Apprill et al., 2015), modified with a linker sequence (Herbold et al., 2015), were used for qPCR amplification of 16S rRNA genes using a CFX96 Real-Time System. The concentration of each sample was adjusted to a concentration between 1-20 ng/ul. PCR assay mixtures consisted of 10ul Brilliant III Ultra-Fast SYBR Green Master Mix already containing a SureStart Taq DNA polymerase, MgCl2 at a concentration of 2.5 mM, two times 0.8 ul primer solution (400 nM), 7.4 ul sterile nuclease-free water, and 1 ul template DNA solution or adjusted 6.4 ul sterile nuclease-free water and 2ul template DNA. The PCR amplification program encompassed an initial denaturation step at 95C for 3 min followed by 40 two-step cycles at 95C for 10 s and at 55C for 30 s. In each run a no-template (without TNA) control was included. Genomic DNA of Escherichia coli DH5alpha was used as a standard (highest concentration 25 ng/ul following a 4 times 1:10 dilution series). 16S rRNA gene amplification and amplicon sequencing was performed at the Joint Microbiome Facility of the Medical University of Vienna and the University of Vienna. The V4 region of the bacterial and archaeal 16S rRNA gene was amplified (30 cycles) from the DNA using the mentioned modified primer pair and barcoded (8 cycles) in a unique dual barcoding setup (UDB-H12). Thereafter, barcoded samples were purified and normalized over a SequalPrep Normalization Plate Kit using a Biomek NXP Span-8 pipetting robot (Beckman Coulter), and pooled and concentrated on columns (Anlaytik Jena). Indexed sequencing libraries were prepared with the Illumina TruSeq Nano Kit as described previously (Herbold et al., 2015), and sequenced in paired-end mode (600 cycles; V3 chemistry) on an Illumina MiSeq following the manufacturers instructions. The workflow systematically included four negative controls (PCR blanks, i.e., PCR-grade water as template) for each 90 samples sequenced.