title: Illumina MiSeq paired end sequencing
design: The study investigates the effect of helminths on the microbial community (eukaryotic and prokaryotic) with 222 samples from 9 rats, plus negative controls.
library_construction_protocol: DNA was extracted from rat fecal samples at the Institute of Parasitology by first homogenizing with the FastPrep-24 instrument (MP Biomedicals, Santa Ana, CA, USA), purified using PSP® SPIN Stool DNA Plus Kit (Stratec Biomedical, Birkenfeld, Germany) according to the manufacturer’s protocol, and amplified using primers and protocols modified from the Earth Microbiome Project. We used redesigned versions of the 515f/806r primers that target the V4 region of 16S rRNA in Bacteria and Archaea: 515f (5’–GTGYCAGCMGCCGCGGTAA–3’), which had 12nt Golay barcode and 806r (5’–GGACTACNVGGGTWTCTAAT–3’) (http://www.earthmicrobiome.org/emp-standard-protocols/16s/). Amplifications were conducted using 25μL reactions and amplified using 5 Prime Hot Master Mix with 1 μL of genomic DNA. PCR conditions: denaturation step at 94°C for 3 minutes, then 25 cycles of 94°C for 45 seconds, 50°C for 60 seconds, and 72°C for 90 seconds, then final extension step at 72°C for 10 minutes. PCR products were visualized on a gel and quantified using Picogreen (Invitrogen) according to the manufacturer’s protocol.  50 ng of each sample PCR product were pooled. The final pool was cleaned using the Ultraclean PCR cleanup kit (MO BIO Laboratories) and sequenced at the University of California in Los Angeles (Genoseq) sequencing core. The pool was sequenced on the Illumina MiSeq platform with paired end 2 x 250 sequencing and a separate 13-nucleotide index read.