Sep 27, 2019

Public workspaceUltra-deep, long-read nanopore sequencing of mock microbial community standards

DOI
dx.doi.org/10.17504/protocols.io.x9tfr6n
  • Josh Quick1,
  • Sam Nicholls1,
  • Nick Loman1,
  • Shuiquan Tang2
  • 1University of Birmingham;
  • 2Zymo Research Corporation
  • Long Read Club
    Tech. support email: n.j.loman@bham.ac.uk
Josh Quick
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DOI: dx.doi.org/10.17504/protocols.io.x9tfr6n
External link: https://www.biorxiv.org/content/biorxiv/early/2018/12/04/487033.full.pdf
Protocol Citation: Josh Quick, Sam Nicholls, Nick Loman, Shuiquan Tang 2019. Ultra-deep, long-read nanopore sequencing of mock microbial community standards. protocols.io https://dx.doi.org/10.17504/protocols.io.x9tfr6n
Manuscript citation:

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 18, 2019
Last Modified: September 27, 2019
Protocol Integer ID: 20499
Keywords: bioinformatics, metagenomics, mock community, nanopore, single-molecule sequencing, real-time sequencing, benchmark, GridION, PromethION, Illumina, de novo assembly
Abstract
Background: Long sequencing reads are information-rich: aiding de novo assembly and reference mapping, and consequently have great potential for the study of microbial communities. However, the best approaches for analysis of long-read metagenomic data are unknown. Additionally, rigorous evaluation of bioinformatics tools is hindered by a lack of long-read data from validated samples with known composition.
Methods: We sequenced two commercially-available mock communities containing ten microbial species (ZymoBIOMICS Microbial Community Standards) with Oxford Nanopore GridION and PromethION. Both communities and the ten individual species isolates were also sequenced with Illumina technology.
Data: We generated 14 and 16 Gbp from GridION owcells and 150 and 153 Gbp from PromethION owcells for the
evenly-distributed and log-distributed communities respectively. Read length N50 was 5.3 Kbp and 5.2 Kbp for the even
and log community, respectively. Basecalls and corresponding signal data are made available (4.2 TB in total).
Results: Alignment to Illumina-sequenced isolates demonstrated the expected microbial species at anticipated abundances, with the limit of detection for the lowest abundance species below 50 cells (GridION). De novo assembly of metagenomes recovered long contiguous sequences without the need for pre-processing techniques such as binning.
Conclusions: We present ultra-deep, long-read nanopore datasets from a well-dened mock community. These datasets
will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and
comparison of current laboratory and software pipelines.
Guidelines
This protocol has been writen for those wishing to reproduce the DNA extractions used to generate the Nanopore sequencing data presented in the publication.
Materials
MATERIALS
ReagentZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
ReagentZymoBIOMICS Microbial Community Standard II (Log Distribution)Zymo ResearchCatalog #D6310
ReagentZymoBIOMICS DNA Miniprep KitZymo ResearchCatalog #D4300
STEP MATERIALS
ReagentZymoBIOMICS Microbial Community Standard II (Log Distribution)Zymo ResearchCatalog #D6310
ReagentZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
ReagentZymoBIOMICS DNA Miniprep KitZymo ResearchCatalog #D4300
Protocol materials
ReagentZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
ReagentZymoBIOMICS Microbial Community Standard II (Log Distribution)Zymo ResearchCatalog #D6310
ReagentZymoBIOMICS DNA Miniprep KitZymo ResearchCatalog #D4300
ReagentZymoBIOMICS Microbial Community Standard II (Log Distribution)Zymo ResearchCatalog #D6310
ReagentZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
ReagentZymoBIOMICS DNA Miniprep KitZymo ResearchCatalog #D4300
ReagentZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300
ReagentZymoBIOMICS Microbial Community Standard II (Log Distribution)Zymo ResearchCatalog #D6310
ReagentZymoBIOMICS DNA Miniprep KitZymo ResearchCatalog #D4300
Safety warnings
Read the specific MSDS documents accociated with the materials used in the protocol.
Before start
Thaw all frozen reagents on ice, mix well and spin down.
Prepare standard
Prepare standard
Transfer Amount75 µL Microbial Community Standard orAmount375 µL Microbial Community Standard II (Log distribution) to a Amount1.5 mL Eppendorf tube.
Note
Each 75 ul of the even community is expected to yield 2000 ng and the log community 220 ng therefore a larger starting volume of the later is required.

ReagentZymoBIOMICS Microbial Community Standard II (Log Distribution)Zymo ResearchCatalog #D6310

ReagentZymoBIOMICS Microbial Community StandardZymo ResearchCatalog #D6300




Retain supernatant
Retain supernatant
Centrifuge the standard at 6,000 xg for Duration00:05:00 before transferring the supernatant to a new Amount1.5 mL Eppendorf
Note
The Microbial Community Standard is shipped in DNA/RNA Shield for safety reasons but this causes lysis of Gram-negative species. Retaining the supernatant prevents unecessary shearing during the subsequent bead-beating step.





Resuspend pellet
Resuspend pellet
Resuspend the cell pellet in Amount750 µL Lysis Solution from the ZymoBIOMICS DNA Miniprep Kit and transfer the resuspended cells to a ZR BashingBead Lysis Tube.
ReagentZymoBIOMICS DNA Miniprep KitZymo ResearchCatalog #D4300




Bead-beat
Bead-beat
Load the lysis tube into a FastPrep-24 5G instrument and bead-beat at 6.0 m/s for 2 cycles of Duration00:00:40 removing the tube and chilling on ice for Duration00:05:00 between cycles.
Equipment
FastPrep-24 5G
NAME
Bead beater
TYPE
MP Biomedicals
BRAND
116005500
SKU
https://uk.mpbio.com/fastprep-24-5g-instrument
LINK



Centrifuge the lysis tube at 10,000 xg for Duration00:01:00 and transfer Amount400 µL supernatant to a Zymo-Spin III-F Filter in a collection tube.

Centrifuge the filter column at 8,000 xg for Duration00:01:00 and transfer Amount400 µL filtrate to a new Amount15 mL Falcon tube.



For the Microbial Community Standard addAmount45 µL of the supernatant from Step 2 and Amount1.485 mL Binding buffer to the tube and mix well. For the Microbial Community Standard II (Log distribution) addAmount225 µL of the supernatant from Step 2 and Amount2.025 mL instead.

Load Amount800 µL onto a Zymo-Spin IIC-Z Column, centrifuge at 8,000 xg for Duration00:01:00 and discard flow through. Repeat as many times as needed to process all the mixture.


Wash and elute the sample as per the ZymoBIOMICS DNA Miniprep Kit instruction manual.
Prepare sequencing libraries using the Ligation Sequencing Kit SQK-LSK109 (Oxford Nanopore Technologies) using Amount1400 ng input DNA and loading Amount50 ng (MinION) or Amount400 ng (PromethION) completed library onto the flowcell.