Merging natural history with

modern analytical workflows…

DNA Barcoding: Introduction

to Front-End Processing

of Biological Specimens

Alex Borisenko, Biodiversity Institute of Ontario

The DNA Barcoding Workflow

BOLD Systems

Web-Accessible Data and

DNA Barcodes

Specimen

Collection Data

Tissue Sample

Photograph

PCR Amplify

Sequence

Extract DNA

3 Components: Collections, Molecular & Informatics

Front-end Processing: The Challenge

Why is this an issue?

Molecular approaches are scaling up...

Collection processing is becoming the

bottleneck and can impede large-scale

DNA barcoding projects

is a major

logistical

challenge! ? Transforming the diversity

of collection management

approaches into standard

lab-compliant format...

Different collections have different standards and traditions…

Search for

compromise

solutions

Help collections

with their needs

Ensure lab-friendly

format

Project utility in non-

barcoding applications

Pre-Lab Processing: The Challenge

Lot-based Sampling

Multiple specimens per lot

Individual specimen tracking with data links to original lot

Barcoding – Specimen-based

One specimen

One tissue sample

One data record

One DNA barcode sequence

Logistical Challenge: Lots and Specimens

Lot

Container with multiple

individuals resulting

from the same

collecting event

Specimen

Collection voucher

representing a single

biological individual

Logistical Challenge: Lots and Specimens

Logistical Challenge: Traditional Collection Management

Attribute: Lot

(multiple unaccounted

specimens; not suitable for

routine barcoding)

Attribute: Specimen

(represents biological individual;

common source for DNA barcodes)

Attribute: Fragment (Skin, skull, tissue, etc.)

(represents part of biological individual)

•Treats characteristics such as „lot‟ or „specimen‟ as storage unit attributes

•Does not establish differential pathways for different types of storage units

•Does not set a framework for fast processing of specimen aggregates

Storage unit

A Typical Collection Management Workflow:

Single sample approach…

Core labs operate in

96-well plate format

Requires compatible

front-end solutions:

NOT SCALABLE!

Logistical Challenge: Scaling Up Molecular Analyses

The Solution: Develop Front-end Buffer Zone!

PRE-LAB PROCESSING CORE LAB PROCESSES

DNA

extraction

PCR

Cycle seq.

gel check

Seq. cleanup

Sequencing

Tissue lysis

Arrayed plate

Imaging

Tissue sampling

/subsampling

Preparation

Databasing

Arraying

Whole vouchers

Label data

Unarrayed

samples

Arrayed tissue

Interfacing Between Collection and Lab

Core Lab

Imaging

LIMS

Collection Management System

Samples Data Images

Logistical independence of operations

Samples are destined for analysis in a core sequencing facility

•Proper vouchering technique to preserve diagnostic

morphology – very taxon-specific

•Specimen numbering convention – to link the samples

with their corresponding specimen records

•Samples need to be preserved in a DNA-friendly fashion

•The nature and quantity of samples has to be compatible

with standard robotic DNA extraction protocols

Barcode Compliance: Specimen Requirements

Data are destined for centralized online repository (BOLD Systems)

Data Collection: BOLD Requirements

MS Excel-based. Submitted by e-mail to BOLD team. 4 pages:

Data Collection: BOLD SpecimenData Spreadsheet

Specimen Imaging: BOLD Image Submission Protocol

Imaging is important!

Retention of an „electronic voucher‟

Darwin Core Triplet REPOSITORY: COLLECTION: catalog number

NOTE2: Catalog number must relate to biological individual (NOT to the lot #!)

Sample ID Field Number Catalog Number Collection Code Institution Storing

MUS COL-000000 Field # or LOT # Museum number Collection Repository name

www.biorepositories.org

NOTE1: Multiple barcode sequences for the same specimens are redundant.

Registry of Biological Repositories

Institutional Acronyms and Collections Codes

Barcode Compliance: Numbering Convention

If your collection is not

registered, please register!

Retain reference to barcode on specimen!

Barcode of Life DNA voucher specimen

Sample ID: MUS SP-00123

BOLD ID: CODE123-09

•Build virtual links to barcode vouchers

•Tag vouchers with barcode information

Barcode Label

Barcode Compliance: Voucher Archiving

Affix

„barcode

label‟

Digital (ideal):

•Collection database

•Field data entry spreadsheets

Data conversion required

Possible data sources:

Analog (not preferred):

•Collection archive

•Field documentation

•Specimen labels

Data digitization required

Barcode Compliance: Provenance Information

Barcode Compliance: Data Management

Other ways to improve data quality...

•Standardize metadata fields for provenance information

that is being collected (compatibility with Darwin Core v.2)

•Taxonomic framework: global checklists, standards for

interim taxonomy, resolution of nomenclatural disputes

•Geographic framework: standard administrative divisions,

agree on transliterations – ISO country and province

codes; decimal coordinates, standard datum (WGS84)

•Agree on taxon-specific extra information to be collected

(relevant to taxonomy or ecological interactions)

DNA-friendly sources:

•Arthropod legs

•Muscle

•Brain

•Gonad (not in insects)

Discouraged sources:

•Guts

•Liver & internal organs

Main features of a barcode-friendly

tissue source:

•Mitochondria-rich

•Low enzymatic activity

•Easy lysis (but not autolysis)

•Low risk of foreign contaminants

Avoid cross-contamination!

DNA-friendly Sampling: Tissue Source

DNA preservation (or degradation) starts during collection

(killing method, exposure to elements, etc.)

Chris Meyer: “Get rid of water & shut down nucleases”

DNA-friendly killing/fixation methods:

•Non-chemical methods (Freezing)

•Ethanol (aquatic, pitfalls and malaise traps)

•Chloroform, Cyanide, Ammonia (insects)

•Isoflurane, carbon dioxide (vertebrates)

DISCOURAGED killing/fixation methods:

•Formalin (marine)

•Ethyl acetate (insects)

•Diluted propylene glycol (malaise traps, pitfalls)

•Most histological solutions

NB! Ensure timely preservation adequate for material

DNA-friendly Collecting: Specimen Fixation

Non-chemical preservation:

•Freezing – ideal, but expensive and logistically difficult

•Drying – good, but sensitive to storage environment

NB! Do not change from one fixative to another!

Chemical preservation (fluid fixation):

•Ethanol – good, common, but has issues

•DMSO, EDTA, SDS – good for DNA, but not morphology

All methods are sensitive to a wide range of factors:

•Nature and quality of tissue

•Quality of fixative/preservative

•Fixation procedure

•Storage conditions

Making Collections DNA-friendly: Preservation

Example: Ethanol Specimen

•Quality (e.g., acidity and additives)

•Reagent concentration (water content)

•Tissue/Ethanol volume ratio

•Relative surface area of sample

•Storage temperature

•Exposure to light

•Fixative evaporation

Example: Dry Specimen

•Drying conditions

•Pretreatment (skin tanning, insect relaxing)

•Ambient humidity

•Storage temperature

•Exposure to sunlight

•Fumigants and preservatives used (PDB, arsenic)

Making Collections DNA-friendly: Other Factors

Specimen Transactions and Data Policies

The need for streamlined and efficient tracking

Specimen Transactions and Data Policies

BMTAClear material transfer protocols (BMTA)

and Transparent data release policy

•How will the materials be stored and analyzed?

•How will molecular data and collaterals be released?

Balance ownership rights and community services

Transform collection specimens into

lab-ready arrays of tissue samples.

Specimen

arraying

Specimen

imaging Data

collection

Tissue

sampling

Front-end Processing: Workshop Highlights

Front-end Processing – from the field to the lab

• Data Collection

• Imaging Procedures

• Sampling Procedures

• Sampling Kits

So, what is that array thing anyway?

Specimen aggregate

matching plate map

12×8 format

95 samples

+ 1 control

Specimen/Sample Arraying: The Concept

D04

Enables batch tracking

through front-end and

curation stages:

•Databasing

•Imaging

•Tissue sampling

•Labelling

Specimen/Sample Arraying: Examples

Subsampling

Direct sampling

Sampling Kits: to Streamline Sample Submission...

Sampling instructions Sampling medium

Microplate

Plant Tube

Rack

Tube Rack

BMTA & Data Policy

Agreement (iBOL)

CD with templates

for data entry

Dispatched by core analytical facilities...

Sampling Kits: What‟s included?

Plate color coding

DNA PCR

Tissue

Sampling Kit Data Package:

• SpecimenData.xls

• ImageData.xls

• CCDB-0000_Record.xls

Plate map–critical component

Cap strips

• MS Excel Platform – familiar interface

• Built-in formulas – data conversion and parsing

• Built-in macros – automation of data transfer

• Tracks the entire chain from field collecting to BOLD

submission and museum deposition (Darwin Core)

Use custom spreadsheet templates for data

tracking and field collection management

Data Collection: Digitize data right in the field!

Expedition or collecting trip

•Broad geographic localization

•Broad time period

•Hosting/responsible organization

•Expedition lead (donor)

A large-scale collecting effort spanning a certain geographic area

May or may not target specific groups or habitats

Diverse suite of collecting methods

•Precise collecting locality (transect)

•Collecting date (or date range)

•Collector(s)

•Habitat(s)

•Method

A small-scale collecting effort targeting a

certain habitat or focal group

Collecting Event

May or may not be successful

Specimen Imaging: Photography Setup

•High image resolution (mp)

•High depth of field

•Even lighting (soft box)

•Efficient processing speed

•Low distortion

•Low diffraction

•Low glare

•Low blur

It's not about the gear!

High image quality can be achieved with

moderate investments – be creative!

•DSLR camera body

•Macro or micro lens

•Macro flash with diffusor (ring)

•Slave flash for backlight

•Opportunistic softbox setup

See video example…

Specimen Imaging: Framing & Orientation

Framing:

• Leave as little margins as

possible!

• Do not “cut off” parts of

specimen

• Scale bar can be useful

Orientation:

• Should display maximum

diagnostic characters

• Should be comparable in a

batch of specimens

• Should be noted in the BOLD

ImageData submission sheet

Specimen Imaging: Background

Black… Or white?

• Clearer outline

• No backlight required

• Good for relief

• Saves toner

• Proximity to background

• Good for translucency

Colour backgrounds are impractical.

Specimen Imaging: Background

Practical Examples: Tool Sterilization

Flame sterilization

(DNA-poor tissue)

• Arthropods

• Plants

ELIMINase sterilization

(DNA-rich tissue)

• Vertebrates

• Large marine invertebrates

NB! Choose the right method!

Conclusion: The “Barcoding Pyramid”

End user High-tech stages

of DNA barcoding

are critically

dependent

on specimen

sourcing

Specimen

sourcing is

dependent

on the

biodiversity

community