Untill now I have always used the Illumina technique for sequencing the V4 region of the 16S rRNA on the Miseq System. Primers targeting the 16S V4 region were designed and tailed with sequences to incorporate Illumina adapters with indexing barcodes, namely 515F/806R (Caporosa et al., 2010).
515 Forward Primer = 5’
AATGATACGGCGACCACCGAG ACGTACGTACG GT GTGCCAGCMGCCGCGGTAA 3’
806 Reverse Primer = 5’
CAAGCAGAAGACGGCATACGAGAT barcode AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT 3’
Recently I have found an application note introducing a protocol for 16S rRNA sequencing targeting the V3 and V4 region, resulting in an amplicon of 459 bp (instead of the 254 bp fragment). As I am interested in the amplicon of 459 bp, I have taken time to investigate the “16S Metagenomic Sequencing Library Preparation” guide. The primers targeting the 16S, V3 and V4 regions are selected from the Klindworth et al. publication (Klindworth et al., 2013).
341 Forward Primer = 5'
TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG CCTACGGGNGGCWGCAG
805 Reverse Primer = 5'
GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG GACTACHVGGGTATCTAATCC
I would like to recuperate most of the primers for the amplification of the 16S V3 and V4 region.
By this I would like to combine the 341 FP (Klindworth et al., 2013) with the 806 RP (Caporosa et al., 2010), but encountered some difficulties.
Is it possible to mutually use the Illumina adapter sequences of 341 FP and 806 RP?
If not, is it possible to link the 341 FP sequence with the 515 FP Illumina adapter sequence?
As for the reverse primers, the sequences are identical, except for the additional 5’G and 3’CC. Are these additional nucleotides of any importance concerning the amplification of the 16S V3 and V4 region?
Your adaptors should look something like this:
Forward: AATGATACGGCGACCACCGAGATCTACAC [forward barcode] ACACTCTTTCCCTACACGACGCTCTTCCGATCT [primer targetting v3]
Reverse: GATCGGAAGAGCACACGTCTGAACTCCAGTCAC [reverse barcode] ATCTCGTATGCCGTCTTCTGCTTG [primer targetting v4]
Foward barcode is optional. Reverse barcode should be 6 bases (reccomend to follow Illumina Truseq LT set.
Set the Miseq sample sheet to Truseq LT.
I suggest cross checking with the Illumina sequence adaptor letter that is publically available.
What difficulties exactly did you encounter? The main hurdle is getting bands on the gel due to the huge size of "adaptor primers".
http://support.illumina.com/content/dam/illumina-support/documents/documentation/chemistry_documentation/experiment-design/illumina-customer-sequence-letter.pdf
Hello,
I have a similar question. Can I use the primers mentioned above (341F and 805R) for 16s amplification, A-tail and ligate them with the Illumina adaptors with indexes hence avoiding any PCR artifacts and perform a Miseq using V2-500 cycle kit?
Hi Ambica,
Yes that is definitely an option. You could buy the individual a-tail, ligation and multiplex PCR modules from NEB to do so.
Another trick to over come the concerns with PCR would be to spike in ~10% of 341F & 805R (without the adaptors) to the primer mix.
Thanks Huan,
Does that just help amplify better? What is the theory behind it?
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