Protocols for metagenomic DNA extraction and Illumina amplicon library preparation for faecal and swab samples

Next‐generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to I...

Full description

Saved in:
Bibliographic Details
Published inMolecular ecology resources Vol. 14; no. 6; pp. 1183 - 1197
Main Authors Vo, A.-T. E., Jedlicka, J. A.
Format Journal Article
LanguageEnglish
Published England Blackwell Publishing Ltd 01.11.2014
Wiley Subscription Services, Inc
Subjects
Animals
Cloaca - microbiology
cloacal
Deoxyribonucleic acid
DNA
DNA - isolation & purification
DNA, Ribosomal - chemistry
DNA, Ribosomal - genetics
faeces/feces
Feces - microbiology
Gene Library
Genes
High-Throughput Nucleotide Sequencing - methods
Metagenomics - methods
microbial ecology
Mouth Mucosa - microbiology
oral
RNA, Ribosomal, 16S - genetics
Sequence Analysis, DNA
solid phase reversible immobilization beads
Songbirds - microbiology
trophic ecology
trophic ecology
oral
microbial ecology
cloacal
faeces/feces
solid phase reversible immobilization beads
Online AccessGet full text

Cover

More Information
Summary:Next‐generation sequencing (NGS) technology has extraordinarily enhanced the scope of research in the life sciences. To broaden the application of NGS to systems that were previously difficult to study, we present protocols for processing faecal and swab samples into amplicon libraries amenable to Illumina sequencing. We developed and tested a novel metagenomic DNA extraction approach using solid phase reversible immobilization (SPRI) beads on Western Bluebird (Sialia mexicana) samples stored in RNAlater. Compared with the MO BIO PowerSoil Kit, the current standard for the Human and Earth Microbiome Projects, the SPRI‐based method produced comparable 16S rRNA gene PCR amplification from faecal extractions but significantly greater DNA quality, quantity and PCR success for both cloacal and oral swab samples. We furthermore modified published protocols for preparing highly multiplexed Illumina libraries with minimal sample loss and without post‐adapter ligation amplification. Our library preparation protocol was successfully validated on three sets of heterogeneous amplicons (16S rRNA gene amplicons from SPRI and PowerSoil extractions as well as control arthropod COI gene amplicons) that were sequenced across three independent, 250‐bp, paired‐end runs on Illumina's MiSeq platform. Sequence analyses revealed largely equivalent results from the SPRI and PowerSoil extractions. Our comprehensive strategies focus on maximizing efficiency and minimizing costs. In addition to increasing the feasibility of using minimally invasive sampling and NGS capabilities in avian research, our methods are notably not avian‐specific and thus applicable to many research programmes that involve DNA extraction and amplicon sequencing.
Bibliography:Museum of Vertebrate Zoology
Department of Integrative Biology at the University of California-Berkeley
istex:BBE983CEF832C03786B8F0D5F746BBAF1228FC04
ArticleID:MEN12269
National Science Foundation - No. DBI-1103583
ark:/67375/WNG-QSGQ7ZD7-C
Appendix S1 Materials and methods. Results from pilot study. Table S1 Literature review of avian studies that have extracted DNA from either faecal or swab samples. Table S2 Avian faecal and swab sample storage and DNA extraction parameters evaluated along with their corresponding performance indices of DNA quality, yield, and PCR success at two genetic loci, microbial 16S rRNA and arthropod COI (where 4 indicates the most robust amplification score) for the comparative pilot study. Table S3 Avian faecal and swab sample DNA extraction parameters evaluated along with their corresponding performance indices of DNA quality, yield, and bacterial 16S rRNA gene PCR success (where 0 indicates no band and 4 indicates the most robust amplification score) for the PowerSoil Kit (MO BIO) and the SPRI-based method. Table S4 Summary of sequencing performance across all three paired-end 250 bp MiSeq runs. Table S5 Recovery of starting material after application of SPRI beads used at three times the sample volume to concentrate amplicons with minimum sample loss as well as after using the concentrated amplicons in the modified with-bead, PCR-free library preparation protocol developed in this study (adjusted for adapter ligation). Table S6 Cost of all supplies and reagents needed to perform the SPRI-based extraction method on avian faecal, cloacal swab, and oral swab samples. Fig. S1 NanoDrop results of metagenomic DNA quality for faecal (A, D), cloacal swab (B, E), and oral swab (C, F) samples extracted using the MO BIO PowerSoil (A-C) and SPRI-based (D-F) methods. Each line represents a different sample. Fig. S2 Amplicon diversity for A) the arthropod COI locus in avian faecal samples extracted using the Zymo Xpedition Fecal/Soil Miniprep Kit and B) the bacterial 16S rRNA locus in avian faecal, cloacal swab, and oral swab samples extracted using the MO BIO PowerSoil Kit and the SPRI-based method developed in this study. Each bar represents a sample, and each color represents a different taxon.
American Vineyard Foundation - No. 2012-1343
ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:1755-098X
1755-0998
DOI:10.1111/1755-0998.12269