TITLE: Using sequins in metagenome experiments.
AUTHORS: Tim Mercer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">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. </div><div class = "text-block">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.</div><div class = "text-block"><span> For further details on the design, validation and use of sequins, we refer users to ‘</span><span style = "font-weight:bold;">Synthetic microbe communities provide internal reference standards for metagenome sequencing and analysis’</span><span> by Hardwick et. al., (2018) </span><span style = "font-style:italic;">Nature Communications.</span><span></span></div></div>

[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.
?. [Laboratory Steps]
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:  ABCDEF1Sample DNA Amount.Resuspension Volume (ddH20)Resuspended Sequins Concentration.Resuspended Sequin Volume to Add to Sample.Mass of Sequins Added.Final Sequin Concentration.210ng1988 ul0.1 ng/ul2 ul0.2 ng2%3100ng188 ul1 ng/ul2 ul2 ng2%4200ng88 ul2 ng/ul2 ul4 ng2%51000ng8 ul10ng/ul2 ul20 ng2%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
ABCDEF1Sample DNA Amount.Resuspension Volume (ddH20)Resuspended Sequins Concentration.Resuspended Sequin Volume to Add to Sample.Mass of Sequins Added.Final Sequin Concentration.210ng1988 ul0.1 ng/ul2 ul0.2 ng2%3100ng188 ul1 ng/ul2 ul2 ng2%4200ng88 ul2 ng/ul2 ul4 ng2%51000ng8 ul10ng/ul2 ul20 ng2%
?. [Laboratory Steps]
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.
?. [Laboratory Steps]
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.]
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.]
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.gzcat ../actinomycete_genomes.fa metasequin_decoy_2.2.fa >actinomycete_decoy.fabwa index -p actinomycete_decoy.bwa actinomycete_decoy.fa
?. [Bioinformatic analysis of .BAM alignments.]
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
?. [Bioinformatic analysis of .BAM alignments.]
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]
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).