SARST-V6 or Serial Analysis of V6 Ribosomal Sequence Tags
SARST-V6 is a procedure to aid in the determination of the composition of microbial communities. Before the original Serial Analysis of Ribosomal Sequence Tagging (SARST) became available there was a direct need for a fast and cost effective method for quantifying and cataloging vast amounts of microbes. Currently, a modified variation, called SARST-V6, is being used for deep sea expeditions and assessments in microbial composition in which many microbial communities are unknown and understudied. Quantifying these deep sea marine microbes is a huge undertaking and sequencing all of the available DNA would be too time consuming and expensive. By using the SARST-V6 method the target of the procedure is the hypervariable V6 section of the 16S rRNA. This focus on the V6 region results in a smaller regions (30-150 base pairs) of amplification during PCR but larger reads after amplification due to the overhangs that occur (discussed below). From these 16S rRNA sequences, the phylogenetic affiliation of microbes can be compared and cataloged.
(Kysela et al., 2005)
The method of the SARST-V6 procedure produces high numbers of concatemers with multiple PCR amplified rRNA tag sequences, specifically from the primers SARST967F and SARST1046R (for 5' to 3'), at the V6 region. This new approach allows for less steps and less waste of enzymes (figure 1). The steps of PCR amplification are repeated heating and cooling steps that repeatedly denature and breaking apart of the DNA. Use of PCR kits (i.e. QIAquick PCR Purification Kit) allows for consistent and reproducible outcomes. These primers are attached to the required sequences and bind to a single strand DNA overhang. This overhang which is produced allows for 30% more read than the original SARST because of the lack of enzymes needed to cut and glue the DNA back together and the ability to add an A-tail to the strand during incubation. Following the amplification process, the removal of extra biotinylated DNA was performed by addition of Bsgl restriction fragments and then purified with an extraction kit. DNA sequencing is performed by use of kits also (i.e. BigDye cycle sequencing kit)
This procedure effectively allows the variability of the rRNA tag sequence to be completely sequenced and compared to other sequences using BLAST and SEALS software. Identification by BLAST and/or SEALS allows for comparison with GenBank.
Above is the pathway of generalized SARST procedure. Much of the process is done with freeware and with use of a linux computer for the eventual goal of calculating a large alpha- and beta- ecological diversity indexes of microbial communities (see Figure 3). These indexes allow researchers to ascertain the total number of microbes within a sample and the precise number of rRNA regions (which indicates abundance). This knowledge has become invaluable for large scale microbial assessments such as the Oceanic Metagenomics Collectionbegun by the J. Craig Venter Institute in 2002 and others.
SARST-V6 is a procedure to aid in the determination of the composition of microbial communities. Before the original Serial Analysis of Ribosomal Sequence Tagging (SARST) became available there was a direct need for a fast and cost effective method for quantifying and cataloging vast amounts of microbes. Currently, a modified variation, called SARST-V6, is being used for deep sea expeditions and assessments in microbial composition in which many microbial communities are unknown and understudied. Quantifying these deep sea marine microbes is a huge undertaking and sequencing all of the available DNA would be too time consuming and expensive. By using the SARST-V6 method the target of the procedure is the hypervariable V6 section of the 16S rRNA. This focus on the V6 region results in a smaller regions (30-150 base pairs) of amplification during PCR but larger reads after amplification due to the overhangs that occur (discussed below). From these 16S rRNA sequences, the phylogenetic affiliation of microbes can be compared and cataloged.
The method of the SARST-V6 procedure produces high numbers of concatemers with multiple PCR amplified rRNA tag sequences, specifically from the primers SARST967F and SARST1046R (for 5' to 3'), at the V6 region. This new approach allows for less steps and less waste of enzymes (figure 1). The steps of PCR amplification are repeated heating and cooling steps that repeatedly denature and breaking apart of the DNA. Use of PCR kits (i.e. QIAquick PCR Purification Kit) allows for consistent and reproducible outcomes. These primers are attached to the required sequences and bind to a single strand DNA overhang. This overhang which is produced allows for 30% more read than the original SARST because of the lack of enzymes needed to cut and glue the DNA back together and the ability to add an A-tail to the strand during incubation. Following the amplification process, the removal of extra biotinylated DNA was performed by addition of Bsgl restriction fragments and then purified with an extraction kit. DNA sequencing is performed by use of kits also (i.e. BigDye cycle sequencing kit)
This procedure effectively allows the variability of the rRNA tag sequence to be completely sequenced and compared to other sequences using BLAST and SEALS software. Identification by BLAST and/or SEALS allows for comparison with GenBank.
Above is the pathway of generalized SARST procedure. Much of the process is done with freeware and with use of a linux computer for the eventual goal of calculating a large alpha- and beta- ecological diversity indexes of microbial communities (see Figure 3). These indexes allow researchers to ascertain the total number of microbes within a sample and the precise number of rRNA regions (which indicates abundance). This knowledge has become invaluable for large scale microbial assessments such as the Oceanic Metagenomics Collectionbegun by the J. Craig Venter Institute in 2002 and others.