title: Nutrition and the gut microbiota in 1018-month-old children living in urban slums of Mumbai, India
design: Samples were DNA-extracted (MoBioDNeasyPowerSoil HTP 96 Kit) per EMP 2018 protocols. Concentration and purity of samples were estimated using the Nanodrop Spectrophotometer and Qubit Fluorometer. Genomic DNA (25 ng) was amplified for 26 cycles using KAPA HiFi HotStart PCR Kit (Kapa Biosystems Inc., Boston, MA USA). The V3-V4 region was targeted for library construction using 341F/806RB primers modified as described previously (107-110). The forward PCR oligonucleotide (50 bp) contained a 5 Illumina sequencing adapter, a 10-nt pad sequence, and the 341 16S specific linker-primer sequence (5-CCTACGGGNGGCWGCAG-3). The reverse PCR oligonucleotide (55 bp) contained the 3 reverse complement of an Illumina sequencing adapter, the 12-nt Golay barcode, a 10-nt pad sequence, and the 16S specific 806R (modified) reverse linker-primer sequence (5-CCGGACTACNVGGGTWTCTAAT-3). The forward and reverse primer concentration (0.2 M each) were analyzed on a 1.2% agarose gel. Round 1 PCR amplicons (1 L, 1:10) were used for Round 2 indexing PCR by amplifying Round 1 PCR amplicons for 10 cycles to add Nextera adaptors (Nextera XT v2 Index Kit, Illumina, USA). Round 2 PCR amplicons were analyzed on 1.2% agarose gel. Amplicons were sequenced on Illumina MiSeq, using 275 bp x 2 paired-end sequences by Genotypic Laboratory (Bangalore, India).