Barcode of life: simple laboratory and analysis workflows for 16S and CO1 analysisGenus- and species-level identification by 16S or CO1 analysis made easy using a rapid laboratory protocol for adapter attachment and new data-analysis workflow

Fig. 1 Laboratory workflow for barcoded 16S and CO1 sequencing

It is often desirable to be able to identify the species present in a complex mixture. This can be achieved by amplifying the bacterial 16S or mammalian cytochrome oxidase (CO1) loci, and comparing the results with a reference database. PCR amplification of specific loci can allow enrichment of the target region in the presence of a large background of other organisms. By modifying the 5’ ends of standard PCR primers used for amplification of these loci we have developed a protocol that attaches our sequencing adapters to the amplicons in approximately 5 minutes, enabling more rapid species identification (Fig. 1).

In the 16S analysis workflow, reads are compared to the NCBI 16S bacterial database using the Basic Local Alignment Search Tool (BLAST), immediately after each read has been basecalled. To validate the workflow, we prepared 1D 16S libraries by PCR amplification of the ZymoBIOMICS Microbial Community DNA Standard, sequenced the libraries on MinIONTM flowcells and passed the basecalled results through the analysis workflow (Fig. 2). We were able to classify all eight bacteria in the mock community to genus level. We calculated the precision at genus level to be 99% for 1D data.

Locus-specific PCR coupled with rapid adapter attachment for 16S and CO1

Analysis workflow and report simplifies amplicon-based species identification

Fig. 2 Analysis report for species identification, shown here for Staphylococcus 16S

Fig. 3 Comparison of whole-genome and 16S identification at a) genus and b) species levels

We generated whole genome and 16S data from the ZymoBIOMICS Microbial Community DNA Standard and compared the number of calls from the WIMP and 16S workflows at the genus (Fig. 3a) and species (Fig. 3b) levels. As expected for a quantitative workflow, the gDNA WIMP calls agree well with the theoretical levels. The identity calls from the 16S data correlate closely with those from the gDNA data, particularly at genus level, but the correct abundance of genera and species is not reflected in the 16S data, possibly due to PCR bias. This is most noticeable for Pseudomonas, to which our 16S primers had mismatches. At species level, the 16S data reveals some false positive calls, reflecting the similarity of the 16S sequences of some genera.

16S compared to whole genome identification at genus and species levels

© 2017 Oxford Nanopore Technologies. All rights reserved.P17001 - Version 4.0

Staphylococcus

Bacillus

Listeria

Enterococcus

Lactobacillus

Salmonella

Escherichia

Shigella

Klebsiella

Enterobacter

Alignment countover 80% accuracy

0 10,000 20,000 30,000 40,000

Selection summary

139,329Reads analysed

139,237Classification

1,320Unique taxa

Staphylococcus

Distribution of alignment accuracies

80 90 1000

1,000

2,000

3,000

Alignment accuracy

Lineage

NCBI Taxonomy ID:

Rank:Average alignment accuracy:

Alignments at this node:Alignments (including child nodes):

1279NCBI organism overviewNCBI taxonomy overviewgenus88.5 %040477

superkingdom:phylum:

class:order:family:

BacteriaFirmicutesBacilliBacillalesStaphylococcaceae

genus: Staphylococcus

16S BLASTN report

a) b)

Salmonella

Escherichia

Pseudomonas

Listeria

Bacillus

Staphylococcus

Enterococcus

Lactobacillus

Saccharomyces

Cryptococcus

0 15 30

16S_1D

gDNA_1D

Relative abundance

0 8 16

S. aureus

B. mojavensisB. subtilis

[Brevibacterium]haloterans

L. innocuaL. welshimeriL. monocytogenes

E. faecalisL. fermentumS. cerevisiaeC. neoformans

S. entericaE. coliP. aeruginosa

Relative abundance

16S_1D

gDNA_1D

The highlighted row is also selected in the Selection summary to the right

16S gene – uses most accurate classification of each read

Species identification – key figures

Top classifications

Fig. 4 DNA extraction, followed by CO1 PCR and library prep on VolTRAX

Sample to result: identification of insect species by CO1 sequencing using VolTRAXIn some situations it is an advantage to be able to identify species outside of a laboratory environment. VolTRAX is a portable device which is designed to perform the necessary steps to convert a raw biological sample to a form ready for analysis on a nanopore sensing device, without the need for human intervention. We extracted DNA from the invasive ladybird species Harmonia axyridis by bead-beating, and loaded the crude extract onto VolTRAX. We performed PCR of a 650 bp region of the cytochrome oxidase gene followed by addition of sequencing adapters using our rapid-attachment chemistry, on VolTRAX, and sequenced the resulting library for 1 hour (Fig. 4a). BLAST analysis of the reads confirmed the identity of the sample (Fig. 4b).

Contact: publications@nanoporetech.com More information at: www.nanoporetech.com and publications.nanoporetech.com

FR

Sample

Resuspensionin buffer

Cell lysis bybead-beating

(e.g. Omnilyse)

Attachment of rapid-sequencing adapters

(5 min)

Bead-washingand elution

Loading

Locus-specific PCRwith barcoded

rapid-attachment primers

Load ontoVolTRAX

Rapid amplicon kit

Performedon VolTRAX

Sequence

a) DNA extraction, PCR and

20-cycle PCR

library preparation

+Bead-beating andDNA isolation

b) BLAST resultsDistribution of the top 100 Blast Hits on 100 subject sequences

Mouse over to see the title, click to show alignments

?

<40 50–80 80–200 > = 20040–50

Query

Color key for alignment scores

1 100 200 300 400 500 600

Description Maxscore

Totalscore

Query cover E value Ident Accession

Harmonia axyridis mitochondrion, partial genome 809 809 99% 0 89% KR108208.1Harmonia axyridis voucher BIOUG<CAN>:TDWG-0189 cytochrome oxidase subunit 1 (COI) gene, partial cds; mito 742 742 92% 0 89% HQ978629.1Harmonia axyridis voucher 08SOCOL-0010 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial 740 740 92% 0 89% KM850971.1Harmonia axyridis voucher BIOUG01807-F08 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial 738 738 92% 0 89% KR482422.1Harmonia axyridis voucher GBOL_Col_FK_0266 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial 738 738 92% 0 89% KM447361.1Harmonia axyridis voucher BIOUG01771-C04 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial 737 737 92% 0 89% KR482435.1Coccinellidae sp. BOLD:AAB5640 voucher BIOUG17323-E07 cytochrome oxidase subunit 1 (COI) gene, partial cds; mito 737 737 92% 89% KR480860.1Harmonia axyridis voucher 08SOCOL-0113 cytochrome oxidase subunit 1 (COI) gene, partial cds; mitochondrial 737 737 92%

0

89% KM849820.10Harmonia axyridis voucher ADU004 cytochrome oxidase subunit I (COI) gene, partial cds; mitochondrial 737 737 92% 89% KC135950.1

Harmonia axyridis mitochondrial partial COI gene for cytochrome c oxidase subunit I, individual 1 737 737 92%

0

89% AM403518.1

0

Query 7

Sbjct 1273

ACATCGTTAAGTATTTTAATTCGG-TAG----ATGAACTAGAGGAAGATTAATTGGCAAC|||||||||||||||||||||||| ||| | |||||||||||||||||||||| |||ACATCGTTAAGTATTTTAATTCGGTTAGAATTAGGAACTAGAGGAAGATTAATTGGAAAC

GACCAAATTTTTA--ATTATTGCGACAGCTCATGCTTTCATTATAATTATCTTTATAGTA|||||||||| || || |||| |||||||||||||||||||||||| |||||||||||GACCAAATTTATAATATAATTGTTACAGCTCATGCTTTCATTATAATTTTCTTTATAGTA

ATAC---TTTTAATTGGGGGTTTT-GAAATTGATTAGTTCC-TTAATAATT-GAGC-CCT|||| || |||||||||||||| |||||||||||||||| ||||||||| |||| |||ATACCTATTATAATTGGGGGTTTTGGAAATTGATTAGTTCCTTTAATAATTGGAGCTCCT

ATCATAAAGATATTGGAACATTATACTTTTTACTTGGAATATGGGGCAGGA--TGTAGGA|||||||||||||||||||||||||||||||| ||||||||| |||||||| ||||||ATCATAAAGATATTGGAACATTATACTTTTTATTTGGAATAT-GGGCAGGAATAGTAGGA 1331

64

Query 65

Sbjct 1332

Query 120

Sbjct 1392

Query 178

Sbjct 1452

119

1391

177

1451

230

1511