Shoreline Complete V1-V9 Kit

Due to the complexity of the barcode-primer sequence and the variabilities in demultiplexing software, we do not suggest combining different library preps in the same sequencing run. 

The following can be pooled together in a single sequencing run. For any other combination, please contact Technical Support at support@shorelinebiome.com.

Yes, we have used the Invitrogen DynaMag™-96 Side Magnet (Cat. No. 12331D) on Lysis-1 tubes with success.

Yes, you can use the Agilent Bioanalyzer. We typically run a minimum of 96 samples therefore it is not cost-effective to use the Agilent Bioanalyzer for large number of samples. However, if you are testing a small number of samples (i.e. 16 samples), you may want to use it.

Shoreline Complete kits are the complete solution. The kit can lyse, purify, and amplify your sample. A separate extraction kit is not required.

Shoreline Wave kits require 10-100 ng of purified DNA.

Bead beating is not required. Shoreline Complete’s novel lysis eliminates the need for bead beating.

To build our primer sets, we performed a search of the SILVA database (hundreds of thousands of complete and partial 16S genes), the Riken database (2,275 complete and 5,664 draft genome sequences including the Human Microbiome Project (HMP) genome sequences data from NCBI), the Athena database which has 18,566 fully assembled genomes from RefSeq database (Dec. 2018).

The databases include organisms from many different sources including human oral, fecal, skin, model organisms, environment, etc. Our design includes all known (sequenced) variants and prospective unknown primer variants based on the rRNA gene structure. We also have a novel primer design that limits the effects of potential primer depletion or competition.

For lysis, purification, and amplification of 96 samples it takes about half a day.

Find the list below for the number of barcodes available per kit.

Yes! Shoreline Biome has the following kits for purchase that support PacBio sequencing.

• The Shoreline Complete V1-V9 kit supports full-length 16S. It provides reagents for lysis, purification, and amplification. This kit yields SMRTbell-ready amplicons of approximately 1,500 bp. The full-length 16S amplicon generally leads to identification of microbes at the species level.
• The Shoreline Complete StrainID kit supports 16S-ITS-23S. It provides reagents for lysis, purification, and amplification. This kit yields SMRTbell-ready amplicons of approximately 2,500 bp. The StrainID amplicon allows identification of microbes at the strain level.
• The Shoreline Wave V1-V9 kit supports full-length 16S. It provides reagents for amplification only. This kit yields SMRTbell-ready amplicons of approximately 1,500 bp. The full-length 16S amplicon generally leads to identification of microbes at the species level.
• The Shoreline Wave StrainID kit supports 16S-ITS-23S. It provides reagents for amplification only. This kit yields SMRTbell-ready amplicons of approximately 2,500 bp. The StrainID amplicon allows identification of microbes at the strain level.

All kits come in 16-tube or 96-well plate format.

Yes! Shoreline Biome has the following kits for purchase that support Illumina 16S sequencing:

• The Shoreline Complete V1-V3 kit provides reagents for lysis, purification, and amplification for sequencing the V1–V3 region of 16S.
• The Shoreline Complete V4 kit provides reagents for lysis, purification, and amplification for sequencing the V4 region of 16S.
• The Shoreline Wave V1-V3 kit provides reagents for amplification for sequencing the V1–V3 region of 16S.
• The Shoreline Wave V4 kit provides reagents for amplification for sequencing the V4 region of 16S.

These kits include Illumina sequencing adaptors which are added to the amplicons during the PCR step. Additionally, both kits output 96 barcode-labeled amplicons ready for Illumina sequencing. All kits come in 16-tube or 96-well plate format.

Currently, our kits do not contain the appropriate primers to amplify fungi or viruses so the V1-V9 and StrainID kits cannot but used for this. We do have ongoing development projects for this application and will update our customers to any new products. Subscribe to our newsletter here.

You will need:

• a magnet for handling the magnetic beads (Invitrogen DynaMag 96 side magnetic rack, #12331D or Magnetic stand-96 #AM10027). Please note that we strongly recommend the Invitrogen DynaMag 96 magnet. We’ve found that users have had the most success with this type of magnet. The Magnetic stand-96 (Thermo Fisher #AM10027) is the only other approved magnet. Plate magnets that have a ‘well’ type magnet rather than a ‘bar’ or ‘post’ will result in low DNA yield.
• for post PCR clean up, the Qiagen MinElute® PCR Purification Kit (Cat. No. 28004).

other required items common to most labs and are listed in each Technical Manual.

Consensus sequence for V1-V3:

• 27f AGRRTTYGATYHTDGYTYAG
• 515r TYACCGCRRCKGCTGGCAC

Consensus sequence for V4:

• 515F GTGRCAGCKGRRGCGGTYA
• 806R GGAYTACNVGGGTHTCTAAK

Consensus sequence for V1-V9:

• 27f AGRRTTYGATYHTDGYTYAG
• 1492r TASVGHTACCTTGTTACCGACTT

Consensus sequence for Strain ID:

• 27f AGRRTTYGATYHTDGYTYAG
• R AGTACYRHRARGGAANGR

Researchers can choose their own microbiome controls. We have several that we’ve used successfully from Zymo and ATCC.

• Process positive control: ATCC MSA-2002, MSA2006, Zymo D6300
• Process negative control: water at the lysis step
• PCR positive control: MSA 1002, MSA1006, Zymo D6305
• PCR negative control: water at the PCR step

Shoreline Biome recommends including controls in your project to monitor lysis and amplification efficiency, as well as environmental contamination.

The Shoreline Complete kits contain a Lyse & Purify sub-kit and an Amplify sub-kit.

The Lyse & Purify Sub-kit contains:

• a 96-well plate or 16 tubes for lysis
• 2 bottles or tubes of DNA purification beads

The Amplify Sub-kit contains:

• a PCR plate seal for amplification
• a 96-well PCR plate containing 96 barcoded primers or 16 tubes containing 16 barcoded primers

For Illumina, these primers target either the V1–V3 or V4 region of the 16S rRNA gene. Following PCR amplification, the amplicons containing the Illumina sequencing adaptor are pooled, purified, and are ready for sequencing.

For PacBio, these primers target either the full 16S rRNA gene or the 16S-ITS-23S region. Following PCR amplification, the amplicons are pooled and ready for the PacBio SMRTbell reaction.

This chart below shows the advantages of the various amplicons. Here, we are looking at 137 E. coli strains. E. coli 16S gene is similar for many strains, so even if we target the full 16S sequence (i.e. V1V9) we would only be able to resolve ~50% of the strains. The StrainID kit allows full resolution and identification of all 137 strains.

We developed the V1-V9 kit for customers who have existing data on the full 16S gene but also want the technological benefits of our Shoreline Biome lysis and primers. In addition, there are a few organisms that cannot be detected with the StrainID kit because the primer targets are not located near one another within the genome. Notably, Helicobacter pylori, Deinococcus radiodurans, some members of Phylum Planctomycetes.

Shoreline Biome kits have been optimized for low-sample inputs. Adding too much sample could cause the assay to fail. The following guidelines are recommended for sample input lysis:

Buffer Considerations: The Shoreline Rapid Prep and Shoreline Complete lysis step is pH dependent, therefore it is important to follow recommended guidelines for sample input. If sample is stored in buffer with pH < 6.5, 10 µL is the maximum input volume. Adding more than this volume can result in suboptimal lysis and affect downstream results.

Coverage is very dependent on experimental design and goals so each project can have different requirements.

Illumina suggests 100,000 reads/sample for 16S analysis. Many researchers have done much lower reads with good results. We have multiplexed 288 samples using Shoreline Biome V4 kits and sequenced on MiSeq with sufficient coverage.

For PacBio sequencing with the StrainID kit we suggest running ~96 samples on one SMRT cell. This gives sufficient reads/sample to identify bacterial strains present at 1% with reasonable confidence on the Sequel I. In general, more reads will yield greater depth per sample, enabling identification of bacteria present at lower levels in the sample. For highly diverse samples where representation below 1% is needed, more than one Sequel I SMRT cell or a single Sequel II SMRTcell may be advisable.

Customers should use “Procedure & Checklist – Full-Length 16S Amplification SMRTbell® Library Preparation and Sequencing” using the SMRTbell Express Template Prep 2.0. 

To find the protocol, go to https://www.pacb.com/applications/complex-populations/microbial/ then expand the Library & Sample Preparation section (“Learn More +”), and click the link to the 16S amplification and sequencing protocol.

Please note that you will be beginning the procedure with an amplicon pool, not individual amplicons as stated in the PacBio protocol. Begin with “AMPure PB Bead Purification” of the already pooled Shoreline PCR products, then skip the PacBio-specific pooling steps, and rejoin the protocol with “SMRTbell Library Construction”. 

Shoreline Complete and Shoreline Rapid Prep kits have been validated on a variety of samples, as shown below. The protocol will vary depending on sample type.

We have had success with the following sample types:

• solid fecal
• liquid fecal, stored in other buffers
• DNA Genotek OMNIgene®•GUT
• tissue Samples
• mouse fecal pellets
• swabs
• saliva
• pelleted cells

For sample types not listed, please contact support@shorelinebiome.com

V1-V3 region is the region targeted by researchers focused on skin and oral microbiome. Find it in our shop here.

Our primers are specific for bacteria and will not amplify other organisms.

Due to the complexity of the barcode-primer sequence and the variabilities in demultiplexing software, we do not suggest combining different library preps in the same sequencing run. 

The following can be pooled together in a single sequencing run. For any other combination, please contact Technical Support at support@shorelinebiome.com.

Yes, we have used the Invitrogen DynaMag™-96 Side Magnet (Cat. No. 12331D) on Lysis-1 tubes with success.

Yes, you can use the Agilent Bioanalyzer. We typically run a minimum of 96 samples therefore it is not cost-effective to use the Agilent Bioanalyzer for large number of samples. However, if you are testing a small number of samples (i.e. 16 samples), you may want to use it.

Shoreline Complete kits are the complete solution. The kit can lyse, purify, and amplify your sample. A separate extraction kit is not required.

Shoreline Wave kits require 10-100 ng of purified DNA.

Bead beating is not required. Shoreline Complete’s novel lysis eliminates the need for bead beating.

To build our primer sets, we performed a search of the SILVA database (hundreds of thousands of complete and partial 16S genes), the Riken database (2,275 complete and 5,664 draft genome sequences including the Human Microbiome Project (HMP) genome sequences data from NCBI), the Athena database which has 18,566 fully assembled genomes from RefSeq database (Dec. 2018).

The databases include organisms from many different sources including human oral, fecal, skin, model organisms, environment, etc. Our design includes all known (sequenced) variants and prospective unknown primer variants based on the rRNA gene structure. We also have a novel primer design that limits the effects of potential primer depletion or competition.

For lysis, purification, and amplification of 96 samples it takes about half a day.

Find the list below for the number of barcodes available per kit.

Yes! Shoreline Biome has the following kits for purchase that support PacBio sequencing.

• The Shoreline Complete V1-V9 kit supports full-length 16S. It provides reagents for lysis, purification, and amplification. This kit yields SMRTbell-ready amplicons of approximately 1,500 bp. The full-length 16S amplicon generally leads to identification of microbes at the species level.
• The Shoreline Complete StrainID kit supports 16S-ITS-23S. It provides reagents for lysis, purification, and amplification. This kit yields SMRTbell-ready amplicons of approximately 2,500 bp. The StrainID amplicon allows identification of microbes at the strain level.
• The Shoreline Wave V1-V9 kit supports full-length 16S. It provides reagents for amplification only. This kit yields SMRTbell-ready amplicons of approximately 1,500 bp. The full-length 16S amplicon generally leads to identification of microbes at the species level.
• The Shoreline Wave StrainID kit supports 16S-ITS-23S. It provides reagents for amplification only. This kit yields SMRTbell-ready amplicons of approximately 2,500 bp. The StrainID amplicon allows identification of microbes at the strain level.

All kits come in 16-tube or 96-well plate format.

Currently, our kits do not contain the appropriate primers to amplify fungi or viruses so the V1-V9 and StrainID kits cannot but used for this. We do have ongoing development projects for this application and will update our customers to any new products. Subscribe to our newsletter here.

You will need:

• a magnet for handling the magnetic beads (Invitrogen DynaMag 96 side magnetic rack, #12331D or Magnetic stand-96 #AM10027). Please note that we strongly recommend the Invitrogen DynaMag 96 magnet. We’ve found that users have had the most success with this type of magnet. The Magnetic stand-96 (Thermo Fisher #AM10027) is the only other approved magnet. Plate magnets that have a ‘well’ type magnet rather than a ‘bar’ or ‘post’ will result in low DNA yield.
• for post PCR clean up, the Qiagen MinElute® PCR Purification Kit (Cat. No. 28004).

other required items common to most labs and are listed in each Technical Manual.

Consensus sequence for V1-V3:

• 27f AGRRTTYGATYHTDGYTYAG
• 515r TYACCGCRRCKGCTGGCAC

Consensus sequence for V4:

• 515F GTGRCAGCKGRRGCGGTYA
• 806R GGAYTACNVGGGTHTCTAAK

Consensus sequence for V1-V9:

• 27f AGRRTTYGATYHTDGYTYAG
• 1492r TASVGHTACCTTGTTACCGACTT

Consensus sequence for Strain ID:

• 27f AGRRTTYGATYHTDGYTYAG
• R AGTACYRHRARGGAANGR

Researchers can choose their own microbiome controls. We have several that we’ve used successfully from Zymo and ATCC.

• Process positive control: ATCC MSA-2002, MSA2006, Zymo D6300
• Process negative control: water at the lysis step
• PCR positive control: MSA 1002, MSA1006, Zymo D6305
• PCR negative control: water at the PCR step

Shoreline Biome recommends including controls in your project to monitor lysis and amplification efficiency, as well as environmental contamination.

The Shoreline Complete kits contain a Lyse & Purify sub-kit and an Amplify sub-kit.

The Lyse & Purify Sub-kit contains:

• a 96-well plate or 16 tubes for lysis
• 2 bottles or tubes of DNA purification beads

The Amplify Sub-kit contains:

• a PCR plate seal for amplification
• a 96-well PCR plate containing 96 barcoded primers or 16 tubes containing 16 barcoded primers

For Illumina, these primers target either the V1–V3 or V4 region of the 16S rRNA gene. Following PCR amplification, the amplicons containing the Illumina sequencing adaptor are pooled, purified, and are ready for sequencing.

For PacBio, these primers target either the full 16S rRNA gene or the 16S-ITS-23S region. Following PCR amplification, the amplicons are pooled and ready for the PacBio SMRTbell reaction.

We developed the V1-V9 kit for customers who have existing data on the full 16S gene but also want the technological benefits of our Shoreline Biome lysis and primers. In addition, there are a few organisms that cannot be detected with the StrainID kit because the primer targets are not located near one another within the genome. Notably, Helicobacter pylori, Deinococcus radiodurans, some members of Phylum Planctomycetes.

Shoreline Biome kits have been optimized for low-sample inputs. Adding too much sample could cause the assay to fail. The following guidelines are recommended for sample input lysis:

Buffer Considerations: The Shoreline Rapid Prep and Shoreline Complete lysis step is pH dependent, therefore it is important to follow recommended guidelines for sample input. If sample is stored in buffer with pH < 6.5, 10 µL is the maximum input volume. Adding more than this volume can result in suboptimal lysis and affect downstream results.

Coverage is very dependent on experimental design and goals so each project can have different requirements.

Illumina suggests 100,000 reads/sample for 16S analysis. Many researchers have done much lower reads with good results. We have multiplexed 288 samples using Shoreline Biome V4 kits and sequenced on MiSeq with sufficient coverage.

For PacBio sequencing with the StrainID kit we suggest running ~96 samples on one SMRT cell. This gives sufficient reads/sample to identify bacterial strains present at 1% with reasonable confidence on the Sequel I. In general, more reads will yield greater depth per sample, enabling identification of bacteria present at lower levels in the sample. For highly diverse samples where representation below 1% is needed, more than one Sequel I SMRT cell or a single Sequel II SMRTcell may be advisable.

Customers should use “Procedure & Checklist – Full-Length 16S Amplification SMRTbell® Library Preparation and Sequencing” using the SMRTbell Express Template Prep 2.0. 

To find the protocol, go to https://www.pacb.com/applications/complex-populations/microbial/ then expand the Library & Sample Preparation section (“Learn More +”), and click the link to the 16S amplification and sequencing protocol.

Please note that you will be beginning the procedure with an amplicon pool, not individual amplicons as stated in the PacBio protocol. Begin with “AMPure PB Bead Purification” of the already pooled Shoreline PCR products, then skip the PacBio-specific pooling steps, and rejoin the protocol with “SMRTbell Library Construction”. 

Shoreline Complete and Shoreline Rapid Prep kits have been validated on a variety of samples, as shown below. The protocol will vary depending on sample type.

We have had success with the following sample types:

• solid fecal
• liquid fecal, stored in other buffers
• DNA Genotek OMNIgene®•GUT
• tissue Samples
• mouse fecal pellets
• swabs
• saliva
• pelleted cells

For sample types not listed, please contact support@shorelinebiome.com

Our primers are specific for bacteria and will not amplify other organisms.