Linking the human microbiome to gastrointestinal disease often requires large sample sizes, so PD0332991 ic50 there is a need for practical specimen acquisition methods that allow analysis of large numbers of human subjects, focusing attention on methods for collecting and analyzing fecal samples. For that reason, we investigated reproducibility within a specimen, effects of storage time and temperature, and effects of lysis and DNA purification methods on the bacterial communities detected. Trends of interest often involve comparisons between individuals, so the variation due to the above factors within a specimen from a single individual was compared to the variation between subjects. We have also compared
methods for 16S rDNA gene amplification and deep sequencing. With issues of sampling and analysis clarified, we are able to reinforce the finding
that human subjects show drastic differences in the compositions of their gut microbiomes. Results Sample acquisition and storage To compare methods for fecal storage and DNA preparation, ten participants were enrolled and studied, of whom 40% were female and 30% were African American (Table 1). Each participant provided a single stool specimen that was sampled multiple times and then used for DNA extraction. Samples were processed selleck screening library immediately (Table 2, condition 8) or were first frozen at -80°C (Table 2, conditions 1-3, 7 and 9), placed on ice for 24 hours and then frozen at -80°C (Table 2, condition 4), placed on ice for 48 hours and then frozen Teicoplanin at -80°C (Table 2, condition 5), or placed in PSP® (Invitek) buffer at room temperature for 48 hours and then frozen at -80°C (Table 2, condition 6). Table 1 Characteristics of participants Total number of participants 10 Female sex 4 Race Black/African-American
3 White 7 Median age (range) 26.5 years (20 – 61) Median body mass index (range) 25.5 (19.2 – 37.4) Current smoker 1 Stool frequency 1-2 times/day 10 Bristol stool category 1 0 2 4 3 1 4 4 5 0 6 1 7 0 Table 2 Sampling methods compared in this study. days at -80C Method Identifier Storage Method DNA Purification Method min max 1 Immediately frozen (-80°C) Qiagen Stool 2 14 2 Immediately frozen (-80°C, sampled 1 cm from sample 1) Qiagen Stool 6 63 3 Immediately frozen (-80°C) MoBio PowerSoil 58 72 4 4C for 24 h, then frozen (-80°C) Qiagen Stool 1 21 5 4C for 48 h, then frozen (-80°C) Qiagen Stool 0 12 6 PSP for 48 h, then frozen (-80°C) PSP 0 12 7 Immediately frozen (-80°C) Qiagen Stool (70°C) 7 7 8 Fresh Qiagen Stool 0 0 9 Immediately frozen (-80°C) Hot phenol with bead beating 118 137 Cell lysis and DNA purification Four methods were used for DNA isolation from stool. Three commercial kits were used to isolate DNA from fecal samples– QIAamp DNA Stool Minikit, PSP Spin Stool DNA Plus Kit, and the MoBio Powersoil DNA Isolation Kit.