Aug 05, 2020
Using sequins in metagenome experiments.
- 1Garvan Institute of Medical Research
External link: http://www.sequinstandards.com
Protocol Citation: Tim Mercer 2020. Using sequins in metagenome experiments.. protocols.io https://dx.doi.org/10.17504/protocols.io.bic3kayn
Manuscript citation:
‘Synthetic microbe communities provide internal reference standards for metagenome sequencing and analysis’ by Hardwick et. al., (2018) Nature Communications..
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
This protocol has been used internally, and by external collaborating laboratories.
Created: July 08, 2020
Last Modified: November 04, 2020
Protocol Integer ID: 39035
Keywords: metagenomics, microbes, synthetic DNA controls, normalization,
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Abstract
Metagenome sequins are a set of synthetic DNA controls that reflect the sequence complexity, GC content, phylogenetic diversity and abundance of a natural microbial community. The sequins are ‘spiked-in’ to your DNA sample, which together undergo to library preparation, sequencing and analysis. The sequins can then be distinguished from you sample DNA in the output library by their synthetic sequence, and analyzed as internal controls.
Sequins are compatible with all standards library preparation and sequencing methods. This protocol describes the laboratory steps required to re-suspend and spike the sequins into your DNA sample, as well as the bioinformatic steps required to analyze sequins in your output library.
For further details on the design, validation and use of sequins, we refer users to ‘Synthetic microbe communities provide internal reference standards for metagenome sequencing and analysis’ by Hardwick et. al., (2018) Nature Communications.
Before start
1. Anaquin Software.
To analyze sequins, we have developed a software toolkit, named Anaquin, that accepts either .FASTQ or .BAM formats, and can be integrate into your bioinformatic pipeline. When anaquin meta processes the .FASTQ or .BAM files, it performs three main functions:
(i) Partition. Anaquin meta will partition the library into smaller sub-libraries comprising either sample or sequin reads/alignments.
(ii) Calibrate. The number or fraction of sequin reads in a library can be modulated. For example, anaquin meta can calibrate the number of sequins reads to comprise 1% of the library (using the –calibrate option). This tool is useful for matching dilutions between multiple replicates and samples.
(iii) Report. Anaquin meta generates several useful reports, including an analysis of library performance (meta_summary.stats), quantitative accuracy (meta_sequins_table.tsv) as well as individual sequin performance (meta_sequins.tsv).
unzip anaquin_3.14.zip
cd anaquin_3.14.0
make
2. Example Data.
To demonstrate the following steps, this protocol uses the following example RNAseq libraries that can be downloaded from https://www.sequinstandards.com/resources/
communityA_metaquin_MixA.R1.fq.gz
communityA_metaquin_MixA.R2.fq.gz
Laboratory Steps
Laboratory Steps
Receiving sequins.
Your sequins should arrive in tubes within a sealed package. Once received, store the sequin tubes at -20C until you are ready to use them.
Preparing sequin stocks.
Meta vector sequins are provided in solution in nuclease-free water at a concentration of 15 ng/μL. Each tube typically contains at least 200ng of sequin DNA (please note that the exact amount may vary).
Please use the table below to guidethe amount and dilution of sequins that should be used according to the sample DNA amount:
| Sample DNA Amount. | Resuspension Volume (ddH20) | Resuspended Sequins Concentration. | Resuspended Sequin Volume to Add to Sample. | Mass of Sequins Added. | Final Sequin Concentration. | |
| 10ng | 1988 ul | 0.1 ng/ul | 2 ul | 0.2 ng | 2% | |
| 100ng | 188 ul | 1 ng/ul | 2 ul | 2 ng | 2% | |
| 200ng | 88 ul | 2 ng/ul | 2 ul | 4 ng | 2% | |
| 1000ng | 8 ul | 10ng/ul | 2 ul | 20 ng | 2% |
For example, we recommend adding sequins to your sample at a 2% fraction by mass, so that approximately 2% of the reads in your output library will be derived from sequins.
Therefore, if the input requirement for your library is 100ng, then you will want to add 2ng of sequin DNA to 100ng of sample DNA to achieve ~2% fraction by mass.
To achieve this, first resuspend the sequins in 200ul of sterile dH2 0 or TE Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0) to reach a ~1ng/ul concentration, and then add ~2ul of this sequin resuspension to your sample
Spike sequins into sample.
Please note that we recommend you confirm the concentration of your sequin dilution before addition to your sample. This can be achieved using a QubitTM or a similar instrument (please note that we have experienced inaccurate quantification of sequins when using NanodropTM).
Once you have added sequins to your DNA sample, the combined sample/sequins mixture is then used as input into your preferred library preparation protocol as per manufacturer’s instructions.
Store remaining sequins.
Once you have re-suspended your sequins, we recommend you store them as single-use aliquots at -20 C to prevent unnecessary future freeze-thaw cycles. Frozen DNA sequins aliquots are stable for at least 6 months. These individual sequin aliquots should then be thawed and added to DNA samples just prior to library preparation.
Analysis of .FASTQ libraries.
Analysis of .FASTQ libraries.
Bioinformatic analysis of FASTQ libraries.
To directly analyze the sequin from your library .FASTQ files, run the following command:
anaquin meta -t 24 -o results --calibrate 0.005 -1 communityA_metaquin_MixA.R1.fq.gz \ -2 communityA_metaquin_MixA.R2.fq.gz
In this example command, we used --calibrate 0.005 to calibrate sequin reads to 0.5% of the NGS library. Results are provided in output directory.
Bioinformatic analysis of .BAM alignments.
Bioinformatic analysis of .BAM alignments.
Build index with decoy chromosome.
Alternatively, users can align their library to a combined index comprising the reference microbial genomes of interest, as well as the sequin decoy chromosomes (chrQ*). The sample reads will thereby align to the microbial genomes, whilst the sequin reads will align to the decoy chromosome.
In this example, we have aligned the library using BWA (Li et. al., 2009) to reference actinomycete genomes.
These reference genome sequences (actinomycete_genomes.fa) can be downloaded from: https://www.sequinstandards.com/resources/)
The user must first build the combined index. We first concatenate the sequin decoy chromosomes to the microbe genome sequences:
gunzip actinomycete_genomes.fa.gz
cat ../actinomycete_genomes.fa metasequin_decoy_2.2.fa >actinomycete_decoy.fa
bwa index -p actinomycete_decoy.bwa actinomycete_decoy.fa
Align FASTQ library to combined index.
bwa mem -t 24 actinomycete_decoy.bwa communityA_metaquin_MixA.R1.fq.gz \ communityA_metaquin_MixA.R2.fq.gz | samtools view -S -b | samtools sort \ >CommunityA_MixA.align.sort.bam
We finally use anaquin meta to analyse the .BAM alignment files.
anaquin meta -t 24 -o results --calibrate 0.005 --combined CommunityA_MixA.align.sort.bam
In this example command, we used --calibrate 0.05 to calibrate sequin reads to 0.5% of the NGS library. Results are provided in output directory (specified using –ouput)
Output Results
Output Results
When anaquin meta is complete, the following files are generated in the output directory:
anaquin.log – Log files recording the usage and execution of sequin processes.
meta_sequin_table.tsv – Quantification of sequins.
meta_sequin.tsv - Detailed report on each individual sequins. meta_ladder_table.tsv – Quantification of synthetic DNA ladder.
meta_ladder.tsv - Sequin alignments/reads derived from the synthetic DNA ladder. meta_sample_* - Sample alignments/reads (excludes sequins).
meta_sequin_* - Alignments/reads derived from sequins.
meta_ladder_* - Sequin alignments/reads derived from the synthetic DNA ladder.
meta_vector_* - Residual alignments/reads derived from sequin plasmid sequences (used during sequin manufacture).