Fig. 1

Study pipeline. a Recruitment of ELBW infants (<1000 g) with no supplementation (AP1E, AP8C, AP5D and AP25D) and ELBW infants with supplementation (P29F, P30N, P31N, P35C) by nurses at the Rosie Hospital (RH) and the NNUH respectively. Term babies (V3 J, V2A) were recruited by researchers. b Optimisation of the bacterial DNA extraction protocol from ELBW infant faeces by testing three different DNA extraction methods (QIAmp DNA Stool Mini Kit, Fast DNA Spin Kit Soil and enzymatic lysis + QIAmp DNA Stool Kit). Bacterial DNA from the study samples was extracted using the Fast DNA Spin Kit Soil and used to prepare three different 16S rRNA gene sequencing libraries. Each library was prepared using a specific pair of primers which target different hypervariable regions (prefixed by a V) of the bacterial 16S rRNA gene: primers 27F-519R target (V1 + V2 + V3), primers 530F-926R target (V4 + V5), and primers 926F-1394R target (V6 + V7 + V8). c A preliminary bioinformatics analysis was performed on two samples using two different bioinformatics pipelines: OTU analysis and the PE protocol. Both bioinformatics approaches were used to compare the different 16S rRNA gene sequencing profiles obtained for the different hypervariable regions tested (V1 + V2 + V3, V4 + V5, and V6 + V7 + V8). (*) Validation of the 16S rRNA sequencing results was performed on three samples (AP8C, P29F and V3 J) by shotgun sequencing