TITLE: Environmental DNA (eDNA) 12S Metabarcoding Illumina MiSeq NGS Protocol with size selection
AUTHORS: Kathleen Pitz, Jacoby Baker

[DESCRIPTION]
This sequencing protocol is intended to directly follow and use the PCR products of the protocol:
"Environmental DNA (eDNA) 12S Metabarcoding PCR Protocol (with Platinum SuperFi II Taq)" which amplifies the hypervariable region of the mitochondrial DNA 12S rRNA gene in eukaryotes. 

This protocol creates a pooled library which is then size selected using a Blue Pippin or Pippin HT to select for the vertebrate/fish band (~350 bp) and remove the co-amplified bacterial band (~435 bp).  Then the pooled product is sequenced on an Illumina MiSeq v2 in a 2x250bp paired end format.

[STEPS]
SECTION: MIOP: Minimum Information about an Omics Protocol
1. 
  
MIOP Term
Value
   
methodology category

 
project

 
purpose

 
analyses

 
geographic location

 
broad-scale environmental context

 
local environmental context

 
environmental medium

 
target

 
creator

 
materials required

 
skills required

 
time required

 
personnel required

 
language

 
issued

 
audience

 
publisher

 
hasVersion

 
license

 
maturity level

  

SECTION: AUTHORS
2. 
  
PREPARED BY All authors known to have contributed to the preparation of this protocol, including those who filled in the template.
AFFILIATION
ORCID (visit https://orcid.org/ to register)
DATE
   
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SECTION: RELATED PROTOCOLS
3. 
  
VERSION
RELEASE DATE This is the date when a given protocol version was finalised
DESCRIPTION OF REVISIONS Please include a brief description of what was changed relative to the previous version
   
1.0.0
yyyy-mm-dd
Initial release
 
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SECTION: ACRONYMS AND ABBREVIATIONS
4. 
  
ACRONYM / ABBREVIATION
DEFINITION
   
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SECTION: GLOSSARY
5. 
  
SPECIALISED TERM
DEFINITION
   
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SECTION: BACKGROUND
6. Summary

This sequencing protocol is intended to directly follow and use the PCR products of the protocol:
"Environmental DNA (eDNA) 12S Metabarcoding PCR Protocol (with Platinum SuperFi II Taq)" which amplifies the hypervariable region of the mitochondrial DNA 12S rRNA gene in eukaryotes. 

The primers (MiFish-U-F & MiFish-U-R) used in the PCR protocol were developed by Miya et al., 2015 for metabarcoding environmental DNA (eDNA) from fishes.

This work was supported by the David and Lucile Packard Foundation, and NASA award 80NSSC21M0032 and NOAA award NA22NOS0120184 in support of the CeNCOOS MBON.
SECTION: BACKGROUND
7. Method description and rationale

This protocol creates a pooled library which is then size selected using a Blue Pippin or Pippin HT to select for the vertebrate/fish band (~350 bp) and remove the co-amplified bacterial band (~435 bp).  Then the pooled product is sequenced on an Illumina MiSeq v2 in a 2x250bp paired end format.
SECTION: BACKGROUND
8. Spatial coverage and environment(s) of relevance

This protocol has been used to sequence extracted DNA from filtered sea water samples taken from marine coastal stations off the western coast of North America (primarily off of California).

sea water [ENVO:00002149]
http://purl.obolibrary.org/obo/ENVO_00002149
SECTION: BACKGROUND
9. PERSONNEL REQUIRED

1 technician
SECTION: BACKGROUND
10. Safety

Identify hazards associated with the procedure and specify protective equipment and safety training required to safely execute the procedure
SECTION: BACKGROUND
11. Training requirements

SECTION: BACKGROUND
12. Time needed to execute the procedure

SECTION: EQUIPMENT
13. 
  
DESCRIPTION e.g. filter
PRODUCT NAME AND MODEL Provide the official name of the product
MANUFACTURER Provide the name of the manufacturer of the product.
QUANTITY Provide quantities necessary for one application of the standard operating procedure (e.g. number of filters).
REMARK For example, some of the consumable may need to be sterilized, some commercial solution may need to be diluted or shielded from light during the operating procedure.
   
Durable equipment




 
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Consumable equipment




 
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Chemicals




 
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SECTION: STANDARD OPERATING PROCEDURE
14. In the following SOP, please use the exact names of equipment as noted in the table above.

Provide a step-by-step description of the protocol. The identification of difficult steps in the protocol and the provision of recommendations for the execution of those steps are encouraged.
SECTION: PREPARATION
15. Follow steps in the protocol "Environmental DNA (eDNA) 12S Metabarcoding PCR Protocol (with Platinum SuperFi II Taq)" through secondary amplification and QC of 12S PCR product.
SECTION: QUALITY CONTROL
24. None
SECTION: BASIC TROUBLESHOOTING GUIDE
25. None
SECTION: REFERENCES
26. None
SECTION: APPENDIX A: DATASHEETS
27. None
SECTION: Size selection of final library
18. 1. After the pooled library was QC’d, the library was size selected with either a Blue Pippin or Pippin HT to select for the vertebrate/fish band (~350 bp) and remove the co-amplified bacterial band (~435 bp).
SECTION: Pool Library
17. 2. The library pools were QC’d and quantified using a combination of Qubit dsDNA HS, Agilent 4200 TapeStation HS DNA1000 and Invitrogen Collibri Library Quantification qPCR assays.
SECTION: Pool Library
16. 1. After secondary PCR, products were run through Invitrogen SequalPrep Normalization Plate (ThermoFisher Scientific) using manufacturer’s protocol to create pooled library.
SECTION: Size selection of final library
19. 2. After size selection, the pooled library was QC’d again to confirm selection of the correct band and new amplicon concentrations.
SECTION: SEQUENCING
20. The pooled product for the genetic locus was loaded on a standard MiSeq v2 flow cell and sequenced in a 2x250bp paired end format using a v2 500-cycle MiSeq reagent cartridge.
SECTION: SEQUENCING
21. 2. The MiSeq run was performed with a 20% PhiX spike added.
SECTION: SEQUENCING
22. 3. Primers complementary to the Fluidigm CS1 & CS2 oligomers were added to appropriate wells of the reagent cartridge to server as sequencing and index read primers.

12S Sequencing primers (5’ to 3’ direction):
FL1-CS1(read1) 
A+CA+CTG+ACGACATGGTTCTACA

FL1-CS2(read2) 
T+AC+GGT+AGCAGAGACTTGGTCT

FL2-CS1rc 
T+GT+AG+AACCATGTCGTCAGTGT

FL2-CS2rc(index) 
A+GAC+CA+AGTCTCTGCTACCGTA

 
  
Sequencing Primer Name
Direction
Sequence (5’ -> 3’)
   
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forward
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reverse
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SECTION: SEQUENCING
23. 4. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0