Hi. two questions: What kit did you use to collect the samples, and have the sequences posted here undergone any QC?
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@mattk is this something you can help with?
If others are interested in the Fox Insight genetic, microbiome, or patient-reported outcome data, it can be found here: Fox Insight: Data Exploration Network.
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Participants received OMNIgene•GUT stool and OMNIgene•ORAL saliva collection kits, with standardized collection instructions. They were instructed to refrain from eating, drinking, chewing gum, smoking, or brushing teeth 30 minutes before saliva collection. Kits contained reagents to rapidly homogenize samples at the point of collection and maintain the DNA stable at ambient temperature for 60 days, with no cold chain required. Participants were instructed to return their samples in prepaid mailing boxes within 2 weeks of collection.
DNA extraction, sequencing, and annotation were performed by Diversigen (Minnesota, USA). Upon receipt of the samples, visual observations of the collection kits were recorded, including sample discoloration, low volume, and incorrect usage. Additional quality controls included determining that there were at least 16,000 16S copies/μL and greater than 1ng/µL of DNA per sample. Microbial DNA was extracted at room temperature from saliva and stool samples using the QIAGEN Powersoil Pro DNA isolation kit with a liquid-handling robot, and according to standardized protocols (QIAGEN, Netherlands). Sequence libraries were prepared using enzymatic tagmentation with low cycle polymerase chain reaction. 88 samples plus a positive and negative library prep control were included in each 96 well-plate. Sequencing was performed with the BoosterShot® shallow shotgun sequencing platform, with 1x100 reads at a target depth of 2 million reads per sample. The Cutadapt software tool was used to discard sequences with an average Q score less than 30. Additionally, samples with fewer than 10,000 reads were discarded.
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If I may add my 2 cents - Diversigen’s BoosterShot is a service rather than a sequencing platform. In their 2018 mSystems publication, they mentioned the Illumina HiSeq as the sequencing technology used in their shallow shotgun sequencing approach.
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To follow up on this, is the annotated sequencing data available for download, or only the raw sequencing files?
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I believe currently just the raw sequencing files. We’ve been in discussion with folks at MJFF to get the OTU and KO tables made available as well in the (hopefully) near future.
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Got it, thanks for letting us know! That would be great if those tables can be made available as well.
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Hi @kiratos, wanted to flag another thread with the availability of annotated data on this thread: Fox Insight PD Microbiome (PDMB) Metadata & Resources. Hope this helps!
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Thanks so much! That’s very helpful.
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