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.



Guidelines for Metagenomic Sequencing (Microbiome) Projects


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



Martin Beer
Friedrich Loeffler Institut - Bundesforschungsinstitut Fuer


Simone Caccio
Instituto Superiore di Sanita