CASE SELECTION:

100 cases of COVID-19 samples proved to be positive in Ain Shams University Specialized
Hospital labs (at the Universities reference lab) will be tested in the strain study.

Detection of virus:

Sample collection:

Nasopharyngeal & oropharyngeal swabs will be taken from suspected cases and put in a specific
viral transpot media.

One oropharyngeal swab (OS) and one nasopharyngeal swab (NS) are collected from hospitalized
SARI patients (adults and pediatric) and then added together in one 15ml Falcon tube
containing 2ml viral transport media (VTM). The swabs are obtained by a well-trained doctor
or medical professional.

Oropharyngeal swabbing: A dry sterile tip flocked with nylon fiber swab applicator is used to
swab both the tonsils and the posterior pharynx. The swab is placed in a 15 ML Falcon tube
labeled with the patient unique ID and containing 2ml VTM (consisting of sterile solution of
bovine albumin fraction V, HEPES buffer, penicillin and streptomycin in HANK's balanced salt
solution). The applicator stick is then cut off.

Nasopharyngeal swabbing: A flexible, sterile tip flocked with nylon fiber swab applicator is
inserted into the nostril and back to the nasopharynx and left in place for a few seconds. It
is then slowly withdrawn with a rotating motion. The swab is inserted in the same tube as the
throat swab and the shaft is cut.

Sample procedures:

The tube containing the nasopharyngeal and oropharyngeal swab will be agitated vigorously for
10 seconds using a vortex mixer. Both swabs should then be removed from the tube and
discarded using sterile forceps that should be sterilized from one sample to another to avoid
cross contamination.

The resulting supernatant (VTM/UTM) is decanted in 2 cryogenic vials labeled with the patient
ID.

One cryogenic vial will be immediately stored at -70°C freezer or lower temperature such as
liquid nitrogen tank for QA and further characterization of the COVID-19.

The other vial will be tested according to the lab capacity:

VIRAL RNA ISOLATION:

Using commercially available kits supplied by thermofisher or other recommended companies.
Work will be done in Biosafety level 2 cabinets under complete sterile conditions.

COVID-19 DETECTION:

Using real time reverse transcription polymerase chain reaction (rRT-PCR) the test can be
done on respiratory samples obtained by various methods, including nasopharyngeal swab or
sputum sample. Results are generally available within a few hours. Molecular methods leverage
polymerase chain reaction (PCR) along with nucleic acid tests, and other advanced analytical
techniques, to detect the genetic material of the virus using real-time reverse transcription
polymerase chain reaction for diagnostic purposes. Commercially available kits supplied by
HVD, thermofisher and others.

Coronavirus Research Using Next-Generation Sequencing (NGS) One assay surveying the entire
coronavirus genome for epidemiological investigation: A major challenge for microbiologists
and virologists is the prediction of patterns of evolution and emergence of disease agents.
RNA viruses like the coronavirus share the biological feature of high genetic variability,
which causes them to appear as clouds of mutants. Coronavirus variants also emerge through
antigenic shift within animal reservoirs, such as bats and snakes. Ion Torrent targeted
next-generation sequencing (NGS) enables a streamlined research workflow for complete genome
sequencing and epidemiological studies of SARS-CoV-2 (the coronavirus responsible for
COVID-19). Through the use of a set of highly specific, universal coronavirus primers in
combination with a high-fidelity master mix, all genomic segments are amplified and the DNA
amplicons are sequenced on any Ion Torrent system to deliver highly accurate coronavirus
typing in under a day.

The Ion AmpliSeq SARS-CoV-2 Research Panel consists of 2 pools with amplicons ranging from
125-275 bp in length for complete viral genome sequencing and variant detection.

Bioinformatic analysis of the NGS DATA Next-generation sequencing, or NGS, can generate
enormous amounts of data, and the challenge becomes finding ways to analyze it properly. In
this study we are going to use some bioinformatic tools to analyze and track the outbreak in
Egypt.

Genome Detective bioinformatic tool, Takes the raw data from the sequencing machine, filter
out results from non-viruses, piece together the genome and use that to identify the virus.
It does not rely on any prior guesses or hypotheses, so it can even identify viruses that
have not been seen before. This was used to confirm the first case of COVID-19 in Belgium,
identifying it as a SARS-related coronavirus.

NextStrain, is an online resource that uses genome data to monitor the evolution of
disease-causing organisms such as viruses in real time. It has tracked several outbreaks
including Zika, Ebola and Dengue and has even been used to inform World Health Organization
policy on seasonal flu.

NextStrain already has over 700 genomes of the new coronavirus, which it can use to trace the
outbreak by detecting new mutations in the virus. The mutations do not necessarily affect how
the virus behaves, but they can act as a genetic signature to link cases that are related.
Like tracing your ancestry through a DNA test, a virus sequenced in Madrid, for instance,
could have mutations that suggest it originated from an outbreak in Italy.

Sequencing cases will become even more important because as we start cracking down on (the
pandemic), which we hopefully will achieve, it will tell us how many transmission chains are
still circulating and whether the virus is being transported from one region to another.

Phylogenetic analysis of these 2019-nCoV genomes and those of other coronaviruses to be
completed by virology consultant

Multiple sequence alignment will be performed with the ClustalW program using MEGA software
(version 7.0.14). Phylogenetic trees will be constructed by means of the maximum-likelihood
method with MEGA software (version 7.0.14). The full-genome viral sequences will be deposited
in the dataset of Global Initiative on Sharing All Influenza Data (GISAISD, No.
EPI_ISL_402123, EPI_ISL_403928-31).