Full-length 16S taxonomic profiling with PacBio
The bacterial 16S ribosomal rRNA is a complex gene that has 9 variable regions. The usual NGS approaches for 16S analysis are based on the sequencing of one or two variable regions of the 16S ribosomal subunit using short reads technologies. Hence, using short read technologies only 2/9 of the variable regions are screened to distinguish the taxa present in a sample.
100% of the hypervariable regions can be analyzed using PacBio reads
Using long read sequencing technologies we can obtain the sequence of the full gene and, then, have a significantly higher specificity and resolution capacity to do the taxonomic assignments based on the differences in the 16S gene sequence.
More specific taxonomic assignments with PacBio
We have developed a specific module of MG7 to analyze the 16S PacBio sequences covering the full gene. We have tested the system using PacBio sequences from BEI mock communities and we have got very good results. In the majority of the cases we got to assign each sequence at the level of strain, the maximum level of specificity.
The application of PacBio sequencing for 16S profiling is possible thanks to the PacBio CCS (Circular Consensus Sequences) mode. This kind of PacBio sequences is accurate enough to allow taxonomic profiling.
To obtain PacBio Circular Consensus Sequences (CCSs) shorter insert sizes are used, giving the polymerase the opportunity to copy several times the same DNA region. These different sequenced copies of the same region, obtained from the same DNA molecule, allow obtaining a corrected consensus sequence with a minimal error rate (final quality around 99.9).
PacBio CSS approach gets high quality long reads from metagenomics samples
Using CCSs it is possible to have several copies of the same DNA molecule. It makes possible to correct the sequences aligning the sequences from the different passes of the polymerase.
See the schema of the PacBio CCS approach:
Schema of PacBio CCS approach
*1: A SMRTbell™ template is a double-stranded DNA template capped by hairpin loops at both ends. The SMRTbell template is structurally linear and topologically circular. Key advantages of the SMRTbell template format include homogeneous, structure of the templates, efficient loading into ZMWs, and complementary, strand information in every molecule. This enables users to apply essentially the same protocol for generating templates of all size ranges. The hairpin adapters that are ligated to each fragment of DNA provide a common primer-binding site for the DNA polymerase.
Definition of SMRTbell™ template taken from Pacbio documentation
Primers for the 16S full gene are more universal
Another advantage of this full-length 16S approach is that the primers are more universal because regions flanking the 16S gene are highly conserved. It allows us amplifying rare bacteria that are not amplified with the usual primers targeted to variable regions.