TITLE: Ultra-deep, long-read nanopore sequencing of mock microbial community standards
AUTHORS: Josh Quick, Sam Nicholls, Nick Loman, Shuiquan Tang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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.</div><div class = "text-block">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. </div><div class = "text-block">Data: We generated 14 and 16 Gbp from GridION owcells and 150 and 153 Gbp from PromethION owcells for the</div><div class = "text-block">evenly-distributed and log-distributed communities respectively. Read length N50 was 5.3 Kbp and 5.2 Kbp for the even</div><div class = "text-block">and log community, respectively. Basecalls and corresponding signal data are made available (4.2 TB in total).</div><div class = "text-block">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.</div><div class = "text-block">Conclusions: We present ultra-deep, long-read nanopore datasets from a well-dened mock community. These datasets</div><div class = "text-block">will be useful for those developing bioinformatics methods for long-read metagenomics and for the validation and</div><div class = "text-block">comparison of current laboratory and software pipelines.</div></div>

[STEPS]
?. [Prepare standard]
Transfer orto a Eppendorf tube.
[Microbial Community Standard]
[Microbial Community Standard II (Log distribution)]
1.5 ml
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.
?. [Retain supernatant]
Centrifuge the standard at 6,000 xg for before transferring the supernatant to a new  Eppendorf
1.5 ml
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 the cell pellet in Lysis Solution from the ZymoBIOMICS DNA Miniprep Kit and transfer the resuspended cells to a ZR BashingBead Lysis Tube.
750 µl
?. [Bead-beat]
Load the lysis tube into a FastPrep-24 5G instrument and bead-beat at 6.0 m/s for 2 cycles of removing the tube and chilling on ice for between cycles.
?. Centrifuge the lysis tube at 10,000 xg for and transfer supernatant to a Zymo-Spin III-F Filter in a collection tube.
400 µl
?. Centrifuge the filter column at 8,000 xg for  and transfer filtrate to a new Falcon tube.
400 µl
15 ml
?. For the Microbial Community Standard addof the supernatant from Step 2 and Binding buffer to the tube and mix well. For the Microbial Community Standard II (Log distribution) addof the supernatant from Step 2 and instead.
45 µl
1.485 ml
225 µl
2.025 ml
?. Load onto a Zymo-Spin IIC-Z Column, centrifuge at 8,000 xg for and discard flow through. Repeat as many times as needed to process all the mixture.
800 µl
?. 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  input DNA and loading or completed library onto the flowcell.
1400 ng
[(MinION)]
[(PromethION)]