The Knight lab has been working hard testing new primers for 16S rRNA amplicon production and its time to share our progress.
So far, the 16S rRNA V4 region forward primer (designated 515f) has been paired with five different reverse primers (806r, 926r, 967r, 1048r, and 1391r) to amplify ribosomal RNA from bacteria, Archaea, and Fungi. Thanks to Jed Fuhrman and Amy Apprill, the 515f and 806r primers have also been modified, helping minimize the amplification bias against Crenarchaeota/Thaumarchaeota (515f) and SAR 11 (806r). All primer pairs have successfully yielded PCR amplicons, and the amplicons from the 515f/806r and 515f/926r constructs sequenced. The remaining primer pair constructs will be sequenced soon with an update to follow once we have the results.
The differences between the old and new 515f nd 806r constructs are described below:
Original 515f construct / modified construct (Jed Fuhrman, C to Y base change on the 5’ end)
5′-GTGCCAGCMGCCGCGGTAA-3′ / 5′-GTGYCAGCMGCCGCGGTAA-3′
Original 806r construct / modified construct (806rB, Amy Apprill, H to N base change mid-primer):
5′-GGACTACHVGGGTWTCTAAT-3′ / 5’-GGACTACNVGGGTWTCTAAT-3’
Why the new constructs, you ask? And what does the added degeneracy mean?
- The barcodes, which were previously located on the reverse primer, are now located on the forward (515) primer. This enables the user to pair the forward primer with various reverse primer constructs to enable longer amplicons. We’ve tested the barcoded 515f primer with 806r and 926r. Importantly, the barcoded constructs were screened in silico for secondary structure against a number of longer constructs (967r, 1048r, 1391r). We have tested the performance of these constructs in PCR but have not validated the results on the MiSeq or HiSeq platforms.
- The degeneracy was added to the forward and reverse primers to minimize the bias against Crenarchaeota/Thaumarchaeota (515f modification) and the marine and freshwater alphaproteobacterial clade SAR11 (806r modification)
To compare the new primer constructs to the old ones and thus confirm the performance of the new constructs, we sequenced amplicons produced from both constructs applied to a number of studies. Our intent was to sample a wide range of sample types to confirm that the new primer constructs produce data comparable to that obtained using the old constructs on a variety of sample types. The studies/sample types that the constructs were tested on are:
-5 American Gut fecal samples
-5 American Gut skin Samples
-5 Body farm control Soil samples
-6 Body farm paired skin/soil samples
-33 Sloan house samples (various sites)
-15 Mouse decomposition control soils
-9 Rice Rhizome samples
-12 Agricultural soil samples
Below is a procrustes plot (using the unweighted UniFrac distance matrix on the left and the weighted UniFrac distance matrix to take into account taxa abundances on the right) comparing samples amplified using the original primer construct and the new, modified primer construct. The calculated M2 value for the unweighted UniFrac based plot is 0.111 and for the weighted UniFrac based plot is 0.196. With the exception of a few mouse decomposition and built environment samples, each sample produces extremely comparable results between the old and new primer constructs. Importantly, very commonly studied sample types (stool, soil, skin) perform very well under the new constructs.