bayesianmice Data Methods Results Code

Primers and barcodes for 16S amplification

16S and ITS amplification.

Using the extracted DNA as a template, primer sequences1,2 containing 10bp unique [barcodes] and 454 FLX Titanium adaptors were used to perform PCR amplification of the internal transcribed spacer (ITS) region of the fungal rDNA and the 16S V3-V5 region of bacteria rDNA.

Barcodes
ACATAGTAGT ACGCACACGT ACGTATGACT ACGTCTCGCT
ACTATATCTA ACTCTAGTCT ACTCTGTGAG ACTGTATATG
ACTGTCTGTA AGACATATCG AGACGTCTCT AGAGCGCGAG
AGAGTACGTG AGAGTCACGA AGATACACAG AGCAGCTACG
AGCGTCAGTA AGTATCTCAG AGTCAGTGTA AGTCGACATG
AGTCTAGTAG AGTCTCTAGA AGTGACTAGT ATACGCACGT
ATACTGCGTA ATATACGCGT ATCGCTAGTA ATCGTAGTGT
CACGATAGCG CAGTGAGACG CATACTACTA CATATAGACG
CGACTACGAG CGCGCACGCG CGCGCTACGT CGTACTCAGT
CGTAGATCGA CGTCAGCACG CGTGAGTGCG CGTGTCGCAG
CTACAGAGAG CTACATCACG CTACGTCTGT CTAGAGTGTG
CTAGCTCGTA CTAGTATGCG CTAGTGCACG CTCACGCGAG
CTCGTGACGT CTCTGTCTCG CTGTACGTAG TACACGATGT
TACGAGTACA TAGCACACTA TAGCGTGATG TAGCTACTGT
TAGTGTAGCG TATACACGCG TATAGCGTCT TATATCGACA
TATCACTACG TATCTGTATA TATGATACTA TCACACTATA
TCACGACGTA TCAGCGTCTA TCAGTCTCGA TCATCGAGTG
TCGACGAGCT TCGACGCATA TCGAGAGTCG TCGAGCACGT
TCGCTACGCG TCGCTATATA TCGCTGATAG TCTACAGCGT
TCTATACGCT TCTATCTAGT TCTCGTGTGT TCTCTCGAGA
TCTGTGACAG TGACGTGACA TGAGACGAGA TGAGATCTCG
TGAGTATGAG TGATGACGCG TGCGTACTCA TGCTACTGAG
TGCTGCGTCG TGTACGCACG TGTACTACAT TGTAGAGTAG
TGTCGTAGCA TGTCTGCTAG


16S Bacterial Primers || V3-V5 Region

Direction Primer Sequence
Forward 341F 5’-CCTACGGGAGGCAGCAG-3’
Reverse 926R 5’-CCGTCAATTCMTTTRAGT-3’


ITS Fungal Primers || Nested Strategy

For fungal ITS amplification, we employed a nested strategy involving an initial outer amplification with the primer set, NSA3 and NLC2, followed by the amplification of the resulting sequences by the primer set ITS1F and ITS4A.3,4,5

Direction Primer Sequence
Nested Forward NSA3 5'-AAACTCTGTCGTGCTGGGGATA-3'
Nested Reverse NLC2 5'-GAGCTGCATTCCCAAACAACTC-3'
Forward ITS1F 5’-CTTGGTCATTTAGAGGAAGTAA-3’
Reverse ITS4A 5’-CGCCGTTACTGGGGCAATCCCTG-3’

Following amplification, the PCR products were cleaned using the Qiagen MinElute 96 UF PCR Purification Kit and quantified using the Invitrogen Quant-DNA Assay Kit, High Sensitivity. 25ng of amplified DNA was pooled from each sample, concentrated and cleaned up to remove any residual primers or nucleotides using the Agencourt AMPure XP Kit. The amplicons were sequenced on the 454 FLX Titanium sequencing platform.

References

  1. Muyzer G, de Waal EC, Uitterlinden AG. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695–700.↩

  2. Lane DJ. 1991. 16S/23S rRNA sequencing. Nucleic acid techniques in bacterial systematics 125–175.↩

  3. Martin KJ, Rygiewicz PT. 2005. Fungal-specific PCR primers developed for analysis of the ITS region of environmental DNA extracts. BMC Microbiol. 5:28.↩

  4. Larena I, Salazar O, González V, Julián MC, Rubio V. 1999. Design of a primer for ribosomal DNA internal transcribed spacer with enhanced specificity for Ascomycetes. J. Biotechnol. 75:187–194.↩

  5. Gardes M, Bruns TD. 1993. ITS primers with enhanced specificity for Basidiomycetes–application to the identification of mycorrhizae and rusts. Mol. Ecol. 2:113–118.↩