Methylation-based enrichment facilitates low-cost, noninvasive genomic scale sequencing of populations from feces

Obtaining high-quality samples from wild animals is a major obstacle for genomic studies of many taxa, particularly at the population level, as collection methods for such samples are typically invasive. DNA from feces is easy to obtain noninvasively, but is dominated by bacterial and other non-host DNA. The high proportion of non-host DNA drastically reduces the efficiency of high-throughput sequencing for host animal genomics. To address this issue, we developed an inexpensive capture method for enriching host DNA from noninvasive fecal samples. Our method exploits natural differences in CpG-methylation density between vertebrate and bacterial genomes to preferentially bind and isolate host DNA from majority-bacterial samples. We demonstrate that the enrichment is robust, efficient, and compatible with downstream library preparation methods useful for population studies (e.g., RADseq). Compared to other enrichment strategies, our method is quick and inexpensive, adding only a negligible cost to sample preparation. In combination with downstream methods such as RADseq, our approach allows for cost-effective and customizable genomic-scale genotyping that was previously feasible in practice only with invasive samples. Because feces are widely available and convenient to collect, our method empowers researchers to explore genomic-scale population-level questions in organisms for which invasive sampling is challenging or undesirable.

. Percentage of reads mapping to the baboon reference genome (papAnu2) for all samples included in this study. Sixteen samples were enriched using the manufacturer protocol and 52 using the revised protocol.    Fig. S1: Percentage of reads mapping to the baboon reference genome (papAnu2) for all samples included in this study. Sixteen samples were enriched using the manufacturer protocol and 52 using the revised protocol.   Table S2: Fecal DNA enrichment results. Key: ID, capture experiment ID; Lib, library ID; Ind, individual (see Supplemental Table S1); PHB, percent host DNA before; TD, total fecal DNA used (ng); BV, bead volume used (µl); TV, total reaction volume (µl); TY, total DNA yield (ng); NE, number of enrichment steps; PHA, percent host DNA after; NDB, n-fold decrease in bacterial DNA. ID Lib  Artificial "fecal" DNA was prepared by manually mixing DNA samples in controlled proportions.

Capture methylated host DNA
Since reaction volumes are well under 100 µl, multiple enrichment reactions can be processed together in a microplate, with pipetting steps conducted using a multichannel pipettor. Compatible rotating mixers and magnetic separators would also be required. Here, we proceed to describe the capture procedure using a 1.5 ml tube.
The total volume of the capture reaction is an important consideration. We have observed decreased DNA binding efficiency when the concentration of MBD beads or DNA in the capture reaction is low. We therefore recommend maintaining a total reaction volume of approximately 40 µl, as we have experienced consistent success with this volume even when adding as little as 1 µl of MBD beads. Decreasing the reaction volume may result in decreased efficacy of rotational mixing. It is a good idea to keep the volume of all reactions consistent as this facilitates processing of many samples and, if DNA amounts and bead volumes are kept consistent, serves as a control for the effects of bead or DNA concentration on enrichment efficiency. Our subsequent procedures assume a reaction volume of 40 µl (not including MBD beads). If using other reaction volumes, pay particular attention to notes following each step in this section.
16. Aliquot 8 µl of 5X bind/wash buffer to a 1.5 ml microcentrifuge tube For reaction volumes other than 40 µl, tune the volume of 5X bind/wash buffer to maintain 1X concentration and adjust accordingly the volume of DNase-free water added in step 17. The volume of MBD beads should be excluded from this calculation as prepared MBD beads are already at 1X concentration.
We recommend equilibrating 5X bind/wash buffer to room temperature prior to aliquoting for more accurate pipetting.
17. Add up to 30 µl of DNA (prepared in step 1) to the tube. Bring the total volume to 40 µl with DNase-free water.
For reaction volumes other than 40 µl, adjust the volume of DNase-free water added to reach the target volume. Be sure to maintain 1X bind/wash concentration.
18. Add MBD beads to the tube using the volume determined in step 2. Pipette the mixture up and down or swirl a few times to mix.
As an approximate rule and as stated above, add 1 µl of MBD beads for every 6.25 ng of target host DNA in each enrichment reaction. If samples contain less than 6.25 ng of host DNA or if the amount of host DNA is not quantified, add 1 µl of MBD beads.
19. Incubate the reaction for 15 minutes at room temperature with rotation. 20. Following incubation at room temperature, briefly spin the tube and place on the magnetic rack for 5 minutes until the beads have collected to the wall and the solution is clear.

21.
Carefully remove the supernatant with a pipette without disturbing the beads. The supernatant is enriched for microbial DNA and may be saved and purified by bead cleanup (Auxiliary protocol A). Otherwise, discard the supernatant.
22. Add 1 ml of 1 bind/wash buffer (kept at room temperature) to wash the beads.
If processing in a microplate, decrease the volume of wash buffer to 100 µl.
23. Carefully remove and discard the wash buffer with a pipette without disturbing the beads.

24.
Optional. Add 100 µl of 1X bind/wash buffer (kept at room temperature) to the beads. Pipette the mixture up and down a few times to mix.
We have found that an additional wash with 100 µl of 1X bind/wash buffer followed by rotation (steps 24 -27) substantially improved enrichment. To skip this wash, proceed to step 28.
25. Mix the beads by rotating the tube in a rotating mixer for 3 minutes at room temperature.
26. Briefly spin the tube and place on the magnetic rack for 2 -5 minutes until the beads have collected to the wall of the tube and the solution is clear.
27. Carefully remove and discard the supernatant with a pipette without disturbing the beads.

Eluting captured host DNA
The NEBNext Microbiome Enrichment Kit includes an elution protocol for captured DNA that includes digestion of DNA-bound MBD beads with proteinase K and elution with TE buffer. We have found that elution with 2 M NaCl is just as effective, is less time consuming, and conserves proteinase K. Most importantly, we have found that DNA samples eluted with 2 M NaCl and purified by bead cleanup can be further enriched in a repeat enrichment reaction. DNA samples eluted with proteinase K and TE buffer and purified by bead cleanup in contrast produced miniscule yields following a repeat enrichment reaction.
28. Add 100 µl of 2 M NaCl (prepared in step 5 and kept at room temperature) to the beads. Pipette the mixture up and down a few times to mix.
If large numbers of samples are being processed, considering lowering the elution volume such that the combined volume of DNA and SPRI beads (see Auxiliary protocol A; step 1) does not exceed the capacity of microplate wells and thereby preclude the ability to parallelize bead cleanups.
29. Mix the beads by rotating the tube in a rotating mixer for 3 minutes at room temperature.
30. Briefly spin the tube and place on the magnetic rack for 2 -5 minutes until the beads have collected to the wall of the tube and the solution is clear.
31. Carefully remove the supernatant to a fresh microcentrifuge tube and discard beads.
32. Proceed to bead cleanup to purify sample (Auxiliary protocol A).

Auxiliary protocols Auxiliary protocol A: Bead cleanup
Portions of this protocol are modified from Pacific Biosciences protocol # 001-252-177-03.