We had fun working on some metagenome projects and here I came up with our standard methods on 16S rRNA-seq and shotgun metagenome sequencing (WGS)!
Qubit (Life Technologies) quantified genomic DNA (12.5 ng each) was subjected to amplify V3 and V4 variable regions of the 16S rRNA gene using a two-step, tailed PCR approach that has been validated by Illumina (Illumina). Locus-specific primers contain sequence tails that allow for a second PCR to add Nextera® XT indexed adapters (Illumina). Tailed primers increase melting point, efficiency, and specificity while avoiding the disadvantages of long primers, such as hairpins, self-dimers, primer dimers, and chimeras.
The sequences of primers used for the first PCR are as follows (both were prepared by IDT DNA Technologies):
- PCR1_Forward (50 bp): 5’–TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG–3’
- PCR1_Reverse (55 bp): 5’–GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC–3’
KAPA HiFi HotStart ReadyMix was used along with 200 nM each of PCR1_Forward and _Reverse primers and thermal-cycled 25 times.
Desired size/amount of the amplicon (~550 bp) was confirmed by a Bioanalyzer High Sensitivity DNA chip (Agilent Technologies) and PCR products were cleaned up by AMPure XP beads (Beckman Coulter). If enough amount of amplicon is not observed, we added 5-10 more PCR cycles and confirmed by a Bioanalyzer High Sensitivity DNA chip again before the AMPure XP bead cleanup. Approx. 0.5 ng of this initial amplicon was subjected to the 2nd PCR using Nextera XT Index Kits (Illumina), which utilizes a dual-index strategy involving 12 (i7) and 8 (i5) indexes, for generating a total of 96 (12 × 8) different index combinations. KAPA HiFi HotStart ReadyMix was used along with 10% (v/v) each of i7 and i5 adapters and thermal-cycled at 8 times.
Desired size/amount of the amplicon (~630 bp) was confirmed by a Bioanalyzer High Sensitivity DNA chip and PCR products were cleaned up by AMPure XP beads. The resulting libraries were further qualified/quantified by Bioanalyzer High Sensitivity DNA chip, qubit (Life Technologies), and Kapa Library Quantification Kit Illumina (Kapa Biosystems). Equimolar libraries were pooled and subjected to Illumina MiSeq sequencing at 2X300 bp (v3 chemistry) supplemented with 5% PhiX spike-in libraries. By sequencing at 2X300 bp on approximately 550 bp amplicons allows a 50 bp of read overlap which can be used for read-stitching feature by MiSeq Reporter software (Illimina Inc.). Illumina CASAVA pipeline Version 1.8 was used to extract de-multiplexed sequencing reads.
WGS (Shotgun metagenomic sequencing)
Qubit (Life Technologies) quantified genomic DNA (1 ng each) was subjected to library preparation using KAPA HyperPlus Library Preparation Kit according to the manufacturer’s instructions. Briefly, DNA was fragmented enzymatically for 10 min at 37°C. 300 nM each of SeqCap Adapter (Roche) was used for adapter ligation and the ligation reaction was conducted at 4°C for overnight. Libraries were amplified with KAPA HiFi HotStart ReadyMix and SeqCap EZ Pre-LM-PCR Oligos 1 & 2 (Roche), using 13 cycles of amplification. Library size and amount were confirmed by a Bioanalyzer High Sensitivity DNA chip and PCR products were cleaned up by AMPure XP beads. If significant amount of larger fragments (> 1000 bp) were observed, we applied SPRI dual size selection (0.5 – 0.7X) to enrich fragments that are between 300 and 500 bp. The resulting libraries were further qualified/quantified by Bioanalyzer High Sensitivity DNA chip, qubit, and Kapa Library Quantification Kit Illumina (Kapa Biosystems). Equimolar libraries were pooled and subjected to Illumina HiSeq 2500 sequencing at 2X100 bp (v3 HiSeq SBS and Cluster Kits (Illumina). Illumina CASAVA pipeline Version 1.8 was used to extract de-multiplexed sequencing reads.
A conference presentation is coming up soon! Good luck, Amanda!!!
Amanda Nelson, Brian Kidd, Yuka Imamura Kawasawa, Joel Dudley, Diane Thiboutot. Isolating and Identifying the Acne Skin Microbiome. Society of Investigative Dermatology Meeting (2016)