Protocols for Sample Processing for NGS

WP 2 in COMPARE was tasked to develop harmonized analytical workflows for generation of high-quality NGS data in combination with relevant metadata for pathogen detection and typing across sample types, pathogens and domains.

Below is a collection of protocols based on those collected from the partners of COMPARE.

The protocols provided below can be either Laboratory Operating Procedures (LOPs) or Standard Operating Procedures (SOPs).

LOPs are those protocols that have been used at least by the provider, but as yet have not been validated in detail.

SOPs are protocols that have been validated in detail and are already published. In some cases, different matrices and different pathogens (such as virus, bacteria, and/or parasite) have been used for validation.

General	Guidelines Guidelines for Metagenomic Sequencing (Microbiome) Projects
Metagenomics	DNA Extraction SOP Isolation of DNA from Fecal and Sewage Samples Using the QIAmp Fast DNA Stool Mini Kit and Modified Protocol LOP Sewages Viral Concentration Using PEG Method LOP Purification of Cryptosporidium Oocysts and Extraction of DNA for NGS Analyses LOP Purification of Giardia cysts and Extraction of DNA for NGS Analyses LOP DNA Extraction Protocol from Chicken Carcass Wash-water LOP DNA Extraction Protocol from Chicken Gut LOP Isolation of DNA from Formalin-fixed and Paraffin-embedded Tissue Using the QIAamp DNA FFPE Tissue Kit
	RNA Extraction LOP Isolation of RNA from Formalin-fixed and Paraffin-embedded Tissue Using the RNeasy Mini Kit LOP Extraction of Total RNA from Concentrated Viral Solution LOP RNA Extraction from Swabs and Tissue Samples
	Pathogen- and Matrix-independent Metagenomics Workflow Harmonized Step-by-step Protocols

Single Isolate	DNA Extraction LOP DNA Extraction (Bacteria) LOP Preparation of Cell Boilates to Extract DNA Suitable for Sequencing Library Preparation
	Library Preparation, including cDNA synthesis and sequencing LOP Sequencing Library Preparation for Illumina Instruments Using the NexteraXT Kit LOP Running Prepared Sequencing Libraries on the Illumina MiSeq Platform LOP RNA Fragmentation, cDNA Synthesis and Library Preparation for NGS Applications LOP Conversion of RNA to Double-stranded cDNA for NGS LOP Agnostic Ion Torrent Sequencing