TITLE: DNA Barcoding Standard Operating Protocol, Plants and Lichens at RBGE, Sanger Sequence Data
AUTHORS: Laura L Forrest, David Bell, Michelle Hart

[DESCRIPTION]
This is part of the collection DToL Taxon-specific Standard Operating Procedures for the Plant Working Group (protocols.io). The SOP collection contains guidance on how to process the various land plant taxa within the scope of the Darwin Tree of Life project. The guidance specifically refers to the tissue samples needed for DNA barcoding (which takes place at the Royal Botanic Garden (RBGE)). Every specimen is submitted for DNA barcoding first before potentially being sent to the Wellcome Sanger institute.

This DNA barcoding SOP outlines the processing of plant and lichen data for the Darwin Tree of Life project (DToL) at the Royal Botanic Garden Edinburgh (RBGE). 

DNA barcoding is used as part of the species identification process AND sample tracking (to check that the genome sequence corresponds to the material that was sent and that there have been no sample mix-ups).

Definition: Land plants (Embryophyta) and lichens
Including: Bryophyta, Marchantiophyta, Anthocerotophyta, Lycopodiophyta, Polypodiophyta, Pinophyta, Cycadophyta, Ginkgophyta, Gnetophyta, Magnoliophyta, lichenized fungi

Excluding: all non-lichenized fungi

[GUIDELINES]

Including: Bryophyta, Marchantiophyta, Anthocerotophyta, Lycopodiophyta, Polypodiophyta, Pinophyta, Cycadophyta, Ginkgophyta, Gnetophyta, Magnoliophyta, lichenized fungi

 



Naming trace files

At RBGE we use an excel sheet to generate trace file names. 
Our DToL Sequencing form concatenates the morphological identification (genus and specific epithet), the DNA number (our EDNA number, which is also the BOLD sample ID), the BOLD primer name and the submitting Genome Acquisition Lab in a standardized format, to be incorporated into the sequence read file name (simplifying the process of deposition data onto BOLD). 

 



Sequence editing

One sequencing has been completed, we upload the reads from each marker and each major lineage (liverwort, moss, hornwort, fern+lycophyte, seed plant) into a separate file for assembly and editing. We use GeneCodes Sequencher software for this. 
Bidirectional reads are assembled by name into contigs, retaining the morphological identification and the BOLD sample ID number as the contig name. Primer sequences are deleted and base calls manually checked. 
Once sequences have been manually edited, the contigs are converted to text, aligned (if coding), the alignment checked for indels or stop codons (if coding), and exported from Sequencher in either aligned (rbcL) or unaligned (ITS, ITS2, psbA-trnH, trnL-trnF) FASTA format. 

 



Verifying sequence data

The FASTA files are checked for laboratory mistakes, contamination issues and major editing errors, using the Basic Local Alignment Search Tool for nucleotide sequence data, BLASTn, against the NCBI database, as well as by comparison to any suitable private databases. This is also useful for spotting any sequences that should be reverse complemented. Potential editing mistakes can be resolved by rechecking the original trace files.
Where there are problems (e.g. fungal sequences generated for ITS2), any trace files that have already been uploaded to BOLD should be flagged as contamination. 
The Genome Acquisition Lab and/or plant collector should be notified of any identification issues that arise with their specimens. 
 



Preparing sequencing data for BOLD

Sequence reads: 
Our FASTA text files require some modification before they can be uploaded to BOLD, to remove the taxon name, and either delete any text in the read name following the BOLD sample ID, or add a pipe character (“|”) directly after the BOLD sample ID.
FASTA files named by the BOLD Sample ID (or the BOLD Process ID) can be uploaded to the relevant BOLD project (for DTOL samples this is the EDTOL project), under the appropriate barcoding marker and sequencing centre (e.g. Royal Botanic Garden, Edinburgh), by copying and pasting the FASTA text. 

Trace Files: 
Once the reads have been manually edited in Sequencher and passed basic quality checks (e.g. that none of the plates have accidentally been reversed so the forwards and reverses don't match...) and primer sequences and low quality regions have been deleted, the SCF 3.0 files for the project can be exported from Sequencher into a new folder, ready to upload to BOLD. 
Using the Terminal or Windows PowerShell and the ls command, get a list of all the SCF file names in the folder. Pasting these into an excel file and using the "convert to text" command is an easy way to extract the BOLD sample ID, primer and locus information from the name of each trace file.
We use a spreadsheet to match BOLD sample IDs with BOLD Process IDs, allowing the BOLD Version 3.0 trace upload form to be completed. 
The trace files are then uploaded to the BOLD EDTOL project in a zipped folder that contains the reads and the completed Version 3.0 form.

 



Passing / failing samples

 


If only one marker has been successfully sequenced:
In most cases the ITS2 marker will be the one which fails (for many reasons including fungal coamplification and microsatellites), and it may not be possible to get a good read for it even if it is re-amplified and re-sequenced. 
When the successfully sequenced marker is adequate to confirm the identity of the taxon (e.g. 100% match to that taxon; other highest hits not found in the UK flora, etc) then the sample can still be considered to have passed barcoding. 

If the taxon is missing from the reference database:
Does it BLAST to something that we would expect to be a close relative? Using the tree-building function in BOLD, does the taxon sit roughly where expected? Then the sample can still be considered to have passed barcoding. 
If the sample is 100% or a very close match to something that is not a close relative of its expected taxon, it can fail barcoding despite the gap in the reference database. 
 


If there is a 100% match to multiple species:
Do these include the expected taxon? If so, the barcode data does not contradict the morphological identification and the sample can be accepted based on the morphological identification.

If there is a 100% match to a different species and a lower match to the expected taxon:
Are the species sometimes synonomised? Taxonomies can vary (e.g. species has been split recently so that older GenBank sequences represent a different species concept, e.g. Conocephalum conicum/Concephalum salebrosum). 
Is the expected taxon sampled from a very different geographic location (e.g. from another continent)? Species concepts are not always applied uniformly geographically, e.g. Metzgeria conjugata in UK and North America. Morphologically similar things on different continents are often given the same name until genetic research shows them to be distinct.
Is the mismatch due to wobbles in either the sample sequence or the reference sequence? (Particularly common for heterozygous or concatenated loci like ITS).
Is there length variation in the reference database, so that a higher similarity can be caused by less variable stretches of the marker being included in the database for a less closely related taxon?

Specimens where the morphology-based identification conflicts with the DNA-barcoding-based identification for any marker should be flagged, lab protocols reviewed for potential contamination issues, and the submitting collector/Genome Acquisition Lab contacted for verification. 
 


Following morphological reverification of flagged samples:
Specimens which still have different morphological and DNA barcoding identifications after the collector/Genome Acquisition Lab has reverified the voucher can be re-extracted from the silica gel dried tissue, after in-house taxonomic review of the silica gel dried tissue for obvious issues (e.g. mixed collections). 
In cases where the silica gel dried tissue appears problematic, DNA extractions may be attempted from the herbarium vouchers instead. 
In cases where the morphological identification of the specimen is revised in line with the DNA barcode data, the specimen in BOLD is edited to reflect the correct taxon name. 
 



Reference databases

UK seed plants: these have been DNA barcoded for two plastid markers (rbcL, matK) and one nuclear marker (ITS2). This data is on BOLD and has been released to NCBI. However, there are some gaps in the dataset, most of which can be filled by a BLASTn search of GenBank.
UK lycophytes: these have been DNA barcoded for three plastid markers (rbcL, matK, psbA-trnH) and one nuclear marker (ITS2). This data is in an open BOLD project. 
UK ferns: For several fern lineages good quality data is available on GenBank (i.e. projects involving well respected taxonomists; projects involving multiple samples per species). 
UK liverworts and hornworts: These have been DNA barcoded for three plastid markers (rbcL, matK, psbA-trnH) and one nuclear marker (ITS2). This data is on private RBGE servers or private projects in BOLD (liverworts and hornworts). For several lineages, good quality data is available on GenBank (i.e. projects involving well respected taxonomists; projects involving multiple samples per species). 
UK mosses: A limited number of UK mosses (e.g. Bryaceae) have been DNA barcoded for three plastid markers (rbcL, matK, psbA-trnH) and one nuclear marker (ITS2). This data is on private RBGE servers. However, there are no UK reference barcode libraries for most UK mosses. In these groups, the well-verified DTOL samples, alongside a small amount of additional sampling, is being used to populate barcode reference libraries, as all DToL material is expert-verified to the highest standards based on morphology. For several lineages, good quality data is available on GenBank (i.e. projects involving well respected taxonomists; projects involving multiple samples per species). 
UK lichens: UNITE is commonly used for fungal barcoding.

 


 





 
     Version      Date      Major changes      Contributors        1.0      2020-04-07      Draft   version      Lyndall   Pereira (NHM)        2.0      2020-04-29      DNA   barcoding for reference library at NHM edited      NHM: Olga   Sivell, Laura Sivess, Gavin Broad, Ben Price, Lyndall Pereira        3.0      2020-05-13      Addition of   pictorial dissection for each taxon group      Liam   Crowley (Oxford WW) and Lyndall Pereira (NHM)        3.1      2020-07-17       SOP text   split into Order-specific SOPs. Uniform style, publication quality images   with colour blind friendly dissection lines      Olga   Sivell, Laura Sivess        4.0      2022-01-04      Overall   formatting further improved (including spelling/ punctuation changes, table   of contents links), photo guides made more uniform, edits in protocol made to   reflect any adaptations in the sampling process since July 2020, additional   taxon protocols included, unrequired taxon guides removed. First submission   to protocols.io.      Inez   Januszczak (NHM), Liam Crowley (Oxford WW), Olga Sivell (NHM), Laura Sivess   (NHM), Chris Fletcher (NHM)   

 

 Working SOP, checked by experts
 

[STEPS]
SECTION: Trace file names
1. Before submitting samples for sequencing, generate trace file names that are in a standardized format as this will facilitate downstream processing. 
 

SECTION: Sequence editing
2. Assemble the forward and reverse traces, trim off low quality areas and primer sequences, and resolve any disagreements between strands.
SECTION: Verifying sequence read and taxonomic identity
4. BLASTn against NCBI to check the top matches (by identity), and look for significant differences in the alignments, e.g. indels. Recheck these against the trace files in your assembly.
SECTION: Putting sequence data onto BOLD
6. FASTA files: rename the sequence identifiers in the definition line to fit BOLD format. Paste the sequences into the BOLD project.
SECTION: Putting sequence data onto BOLD
7. Trace files: Export the trace files in SCF format, fill in the BOLD spreadsheet, and upload the files and spreadsheet to BOLD in a zipped folder.
SECTION: DNA barcode check
8. Check ID using a mixture of BLASTn against NCBI, BOLD taxon ID trees and Full DB searches, and comparisons with in-house reference databases.

 

SECTION: Sequence editing
3. Export the sequence reads in FASTA concatenated format.
SECTION: Verifying sequence read and taxonomic identity
5. Export the revised sequence reads in FASTA concatenated format.
SECTION: DNA barcode check
9. Inform the collector/Genome Acquisition Lab of any samples that are not a good match to the expected taxonomy so that they can check the voucher specimen and photographs.
SECTION: DNA barcode check
10. Update taxon names on BOLD to reflect the most accurate current identification of the sample.