DETECTION OF NEW VARIANTS COVID-19 FROM

PURWOKERTO, BANYUMAS, CENTRAL JAVA

RESEARCH PROPOSAL

Adhelia Intan Purnamasari

B1B018029

MINISTRY OF EDUCATION, CULTURE, RESEARCH, AND

TECHNOLOGY
JENDERAL SOEDIRMAN UNIVERSITY
BIOLOGY FACULTY
PURWOKERTO

2022
 DETECTION OF NEW VARIANTS COVID-19 FROM
PURWOKERTO, BANYUMAS, CENTRAL JAVA

Adhelia Intan Purnamasari

B1B018029

Proposed as a guideline for the implementation of bachelor thesis

in the Faculty of Biology, Jenderal Soedirman University
Purwokerto

APPROVED ON

Supervisor, Co-supervisor,

Dra. Endang Srimurni K., S.U, Ph.D. Dr. Daniel Joko Wahyono, M.Biomed
NIP 195802241983032001 NIP 196309131991031003

Acknowledged by,
Vice Dean for Academic Affairs the Faculty of Biology
Jenderal Soedirman University

Dr. Hendro Pramono, M.S.

NIP 195907221986011001

ii
 PREFACE

Praise Allah SWT, who has given His grace and guidance so that the author to
accomplish this research proposal as a guideline for my bachelor thesis at the Faculty
of Biology Jenderal Soedirman University. The topic is "Detection of New Variants
COVID-19 From Purwokerto, Banyumas, Central Java".
This topic falls within the scope of Virology. To obtain a Bachelor of Science
degree at Jenderal Sudirman University, on this occasion, the author would like to
thank Dr. Hendro Pramono, M.S. as The Vice Dean of Academic Affairs Faculty of
Biology Jendral Soedirman University for his permission to conduct the research.
Notable appreciation author addressed to Dra. Endang Srimurni Kusmintarsih, S.U,
Ph.D., and Dr. Daniel Joko Wahyono, M.Biomed. as a supervisor and co-supervisor
respectively for their contribution in giving me direction in the preparation of this
proposal and Drs. Edi Basuki, Ph.D. as Academic Supervisor, has guided and given
support author at Faculty of Biology Jendral Soedirman University. The author also
thanked all the parties who helped and supported the author in completing the
research proposal, which cannot be mentioned one by one.
The author realizes the limitation of this proposal. Therefore, the
author welcomes any criticism and suggestion to improve this proposal. Hopefully,
this thesis proposal can be used as a guideline in conducting research.

Purwokerto, 2022

Adhelia Intan Purnamasari

iii
 TABLE OF CONTENTS

PREFACE...................................................................................................................iii
TABLE OF CONTENTS............................................................................................iv
LIST OF FIGURES......................................................................................................v
LIST OF TABLES.......................................................................................................vi
SUMMARY...............................................................................................................vii
I. INTRODUCTION.................................................................................................1
II. LITERATURE REVIEW......................................................................................3
III. MATERIALS AND METHODS.....................................................................6
A. Materials, Location, and Time of Research......................................................6
1. Materials.......................................................................................................6
2. Location and Time of Research....................................................................6
B. Research Methods.............................................................................................6
1. Research Design...........................................................................................6
2. Research Parameter......................................................................................7
3. Research Procedures.....................................................................................7
C. Research Framework......................................................................................18
OBSERVATION FORM............................................................................................19
IV. RESEARCH SCHEDULE..............................................................................20
REFERENCES...........................................................................................................21

iv
 LIST OF FIGURES

Figure 3.1 Pangolin COVID-19 Lineage Assigner ‘Drop Zone’...............................15

Figure 3.2 Pangolin COVID-19 Lineage Assigner ‘Start Analysis’.........................15
Figure 3.3 Pangolin COVID-19 Lineage Assigner ‘Result Review’.........................16
Figure 3.4 Visualized the geographical and temporal distribution in Microreact......17

v
 LIST OF TABLES

Table 3.1 Settings for PCR Examination......................................................................8

Table 3.2 Components for cDNA Preparation.............................................................9
Table 3.3 Requirement for Incubation..........................................................................9
Table 3.4 Component for Q5 Hot Start High-Fidelity 2X Master Mix........................9
Table 3.5 Thermal Cycler Program for Thermomix The Reaction............................10
Table 3.6 Component for PCR Dilution.....................................................................10
Table 3.7 Component for Master Mix of End-preparation.........................................10
Table 3.8 Component for Native Barcoding Reaction...............................................11
Table 3. 9 Component for Sequencing of Library Dilution........................................13
Table 3. 10 Samples Extraction Result.......................................................................19
Table 3. 11 Proceeds Samples for Whole-Genome Sequencing................................19

vi
 SUMMARY

In December 2019, a pneumonic outbreak caused by the virus existed in

Wuhan, Hubei province. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-
CoV-2) inducing Covid-19 is occurring infectious diseases nowadays. Covid-19 was
assessed as a public health emergency of international concern (PHEIC) on January
30, 2020. Then, it was indicated as a pandemic by the WHO on March 11, 2020. On
March 2, 2020, the first case of Covid-19 in Indonesia was discovered. On March 21,
2020, the number of positive COVID-19 patients was 450 people, whereas, on that
date, the first COVID-19 case in Purwokerto was confirmed by the Regent of
Banyumas, Achmad Husein. Based on the situation, it is arresting to know the
genetic variation because there has been no detailed report on which genetic
variation spreads in Purwokerto, Banyumas. Therefore, this study aims to understand
the new variant of Covid-19 spreading in Purwokerto, Banyumas, Central Java based
on the extraction and sequencing processes of the samples collected from the
respondents infected with Covid-19 viruses in the Purwokerto on March 25, 2021;
April 1, and 9 2021. The collected samples that have been sequenced will be
identified using Whole-genome sequencing (WGS).
Keywords: COVID-19, genetic variant, Purwokerto, sequencing

vii
 I. INTRODUCTION

In late December 2019, an outbreak of a novel coronavirus disease 2019

(COVID-19) originated in Wuhan, China. A new strain of betacoronavirus
compelled it: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
(Sekizuka et al., 2020). Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-
CoV-2) causing Covid-19 is one of the most significant devastating emerging
contagious diseases nowadays. Covid-19 was typed as a Public Health Emergency of
International Concern (PHEIC) on January 30, 2020. Then, it was portrayed as a
pandemic by the WHO on March 11, 2020. Several aspects were experienced in the
occurrence of the Covid-19 pandemic. Firstly, the global spread of the new SARS-
CoV-2 (without prior human exposure or immunity). Secondly, SARS-CoV-2 as an
RNA virus shows high mutation rates that allow immediate diversification at the cost
of developing non-viable descendants. The virus transmission rate or basic
reproduction number (R0) is high. Furthermore, up to date, no adequate treatment
nor vaccine exists for Covid-19 (Ibrahim, 2020).
Indonesia's hot tropical climate and high UV light intensity are believed to be
sufficient to interfere with the survival and spread of SARS-CoV-2 in the air. This
belief is supported by the situation of the first SARS epidemic, where the cases were
lower than the outbreak. Because this virus originated in China, before March 2020,
Indonesia only carried out individual tests with symptoms and a history of travel
outside the city or country (Cahyani et al., 2021). On March 21, 2020, the number of
positive COVID-19 patients was 450 people, whereas, on that date, the first COVID-
19 case in Purwokerto was confirmed by the Regent of Banyumas, Achmad Husein.
As of today, more than a year after Indonesia’s first COVID-19 case was
announced (i.e., early-March 2020), it is thought that the number of cases in
Indonesia is still significantly underestimated. Besides the utility of screening and
tracing for containing and reducing transmission, there is also a need to pinpoint and
follow variants of SARS-CoV-2 that might worsen the pandemic. World Health
Organization defines a variant to be a Variant of Interest (VoI) if it has changed
phenotypically from the reference genome (or shown phenotypic implications) and
has been identified to cause community transmissions or detected in multiple
countries. A VoI can become a Variant of Concern (VoC) if it has been demonstrated
to 1) increase transmissibility or detrimental changes in COVID-19 epidemiology; 2)

1
an increase in virulence or change in clinical disease presentation; or 3) decrease in
the effectiveness of public health and social measures or available diagnostics,
vaccines, and therapeutics (Cahyani et al., 2021)
SARS-CoV-2 is constantly mutating as an RNA virus, shifting from the
original SARS-CoV-2 viral genotype WIV 04 (hCoV-19/ Wuhan/WIV04/2019) to
several variants found across the world. COVID-19 variants such as the Alpha
(B.1.1.7), Beta (B.1.351) and Delta (B.1.617.2), and the B.1.466.2 variant are
originally hosted by Indonesia. From the end of 2020 to early 2021, genomic
surveillance reported that a significant portion of COVID-19 in Indonesia had
already shifted from the wild-type WIV 04 to other variants, most notably the
B.1.466.2 variant. Two mutations correlated to increased infectivity are normally
found in the B.1.466.2 variant: N439K (99.1%) and P681R (69.7%). The Delta
(B.1.617.2) variant became the predominant variant in Indonesia. World Health
Organization (WHO) classified the Delta variant as a variant of concern (VoC) due
to its potential ability to decrease vaccine effectiveness, increased risk of
hospitalization, and its increased transmissibility (as shown by its raised basic
reproduction number (R0)) (Tenda, et al., 2021).
Some critical understanding of the development of Covid-19 variants were
explored in this study. The benefits that obtained from this research are expected to
provide scientific information regarding the Covid-19 variants develops during the
pandemic in Purwokerto, Banyumas. The results of this research can be used as the
basis for the development of Covid-19 variants based on the date of the samples
taken in Purwokerto, Banyumas.
This research aims to know the new variant of COVID-19 spreading in
Purwokerto, Banyumas, Central Java.

2
 II. LITERATURE REVIEW

Coronaviruses (CoVs) are enveloped viruses with single-stranded positive-

sense RNA belonging to the subfamily Coronavirinae in the family Coronaviridae
(order Nidovirales). CoVs are classified into four genera: Alphacoronavirus,
Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. CoV genomes range
from 25 to 32 kb and have high genetic diversity. CoV is associated with upper and
lower respiratory disease or gastroenteritis in mammals and birds. In humans, CoVs
infection is generally caused by HCoV-229E and HCoV-OC43, which can cause
mild respiratory disease. A new CoV that causes severe acute respiratory syndrome
(SARS-CoV) emerged in humans in 2002-2003 and infected more than 8,000 people
with an evaluated mortality rate of around 10%. The emergence of SARS-CoV with
a mortality rate can induce the risk of a new pandemic threatening public health.
(Razanajatovo et al., 2015).
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a
response to the coronavirus disease (COVID-19). SARS-CoV-2 emerged and spread
globally in 2020, causing a huge pandemic still ongoing today (Gand et al., 2021).
Mercer & Salit (2021) stated that On January 7, 2020, the novel coronavirus was
detected through metatranscriptomic sequencing of lung fluid from a patient with
pneumonia-like symptoms in Wuhan, China. On January 10, 2020, the genome of the
collected sample called severe acute respiratory syndrome coronavirus 2 (SARS-
CoV-2) was published. Within 2 weeks, the first diagnostic tests to detect the virus
were issued. To date, hundreds of millions of people have been tested for the
existence of SARS-CoV-2, leading to widespread public awareness and debate
regarding diagnostic concepts and technologies.
SARS-CoV-2 has a high replication rate. Therefore, viral variants can
influence pandemic dynamics, sequencing the SARS-CoV-2 genome being a
practical tool in predicting outbreaks. WHO publishes guidelines that underlie the
importance of establishing early national surveillance using genome sequencing.
These guidelines highlight the importance of tracking variants to identify if variants
are potentially more difficult to manage quickly. (Tenda, et al., 2021). Cahyani., et al
(2021) supported with more explanation. These variants' existence and potential
emergence highlight the need for reliable, easy-to-regulate, fast and cost-effective
genome sequencing technology in Indonesia. On a small number of samples, they

3
demonstrated how the Oxford Nanopore Technology® (ONT) platform could be
used for whole-genome sequencing (WGS) of SARS-CoV-2 in the Indonesian
context.
Whole-genome sequencing of viruses that play a role in outbreaks,
epidemics, and pandemics is necessary for preventing and reducing the spread of
disease. Whole-genome sequencing can brief epidemiological scientists about
viruses' nature, behavior, and transmission patterns. This information can support
decision-makers in taking suitable action. In the era of next-generation sequencing
(NGS), WGS for epidemiological monitoring should be straightforward, in principle,
with the availability of multiple tools and relatively low sequencing costs compared
to a decade or two ago. Some examples of WGS using these NGS principles in (or
after) outbreaks are the Zika virus in Brazil, Ebola in West Africa, and, more
recently, the mumps outbreak in Canada. Whole-genome sequencing is also at the
heart of global research for vaccine and treatment discovery for the COVID-19
pandemic and surveying its VoC. As part of the SARS-CoV-2 WGS, recording
sample metadata is important for building a clearer picture of genomic epidemiology.
Indonesia has successfully recorded and shared its genomic metadata on GISAID,
despite the sporadic origin of the samples. Metadata information should be made
available and accessible to selected relevant parties (e.g., ministries of health,
medical professionals, and epidemiological researchers) (Cahyani et al., 2021).
Additionally, 2 million SARS-CoV-2 genome sequences have been
developed and shared since the start of the COVID-19 pandemic. They are an
essential basis of information that declares outbreak control, disease surveillance,
and public health policy. Pango dynamic nomenclature is a system for classifying
and naming genetically distinct SARS-CoV-2 lineages, including the variants of
interest. It is based on complete or near-complete analysis of viral genomes. The
Pango dynamic nomenclature system was developed and published in early 2020
(https://pango.network) and has since become a widely used tool worldwide for
classifying SARS-CoV-2. Each Pango lineage aims to define an epidemiologically
suitable phylogenetic group, e.g. introduction to a different geographic area with
evidence of subsequent transmission, the reappearance of previously observed
lineages, or rapid growth of lineages with major phenotypes. Pango lineage is a fine-
scale phylogenetic label designed for outbreak investigations on a national or
regional scale. Therefore, the Pango nomenclature includes many lineages (currently

4
> 1300; https://cov-lineages.org) covering the full genetic diversity of SARS-CoV-2,
some of which are genetically very similar to each other. In contrast, the 'Greek
letters' system recently submitted by the WHO Virus Evolution Working Group is
intended for public communication purposes and labels only a small number of
variants of concern (VOC) and interest of variant (VOI). The WHO variants of the
attention label Alpha, Beta, Gamma and Delta correspond to the Pango lineages
B.1.1.7, B.1.351, P.1, and B.1.617.2, respectively (O’Toole et al., 2022).

5
 III. MATERIALS AND METHODS

A. Materials, Location, and Time of Research

1. Materials

The materials that will be used include lysis buffer, proteinase-K,

elution buffer, alcohol absolute, washing buffer A, washing buffer B, binding
columns, collection tube, Eppendorf tube with 1,5 ml volume, aerosol barrier
tips of 1000 µl, 200 µl, 100 µl, 20 µl and 10 µl, super mix, enzyme mix,
positive control (PC), negative control (NC), internal control (IC). ARTIC
nCoV-2019 V3 panel (100uM), LunaScript RT SuperMix Kit, Q5 Hot Start
HF Polymerase or Q5 Hot Start High-Fidelity 2X Master Mix, dNTP
Solution Mix (10 mM ea.), Nuclease-free water (100 mL), NEBNext Ultra II
End Repair/dA-tailing module, Blunt/TA Ligase Master Mix, Native
Barcoding Expansion Kit 1-12 and/or, Native Barcoding Expansion Kit 13-24
or Native Barcoding Expansion Kit 96, AMPure XP beads , NEBNext Quick
Ligation Module, Sequencing Auxiliary Vials, Short Fragment Buffer
Expansion Kit, Qubit dsDNA HS Assay Kit, Flow Cell Priming Kit, Flow
Cell Wash Kit (optional), and R9.4.1 flow cells
The tools that will be used include vortex, centrifuge, micropipette
with 1000 µl, 200 µl, 100 µl, 20 µl and10 µl volumes, qRT-PCR, Biological
Safety Cabinet (BSC) II, MinION
2. Location and Time of Research

The research will be conducted in the Laboratorium Riset Terpadu of

Jenderal Soedirman University and LIPI Cibinong from September 2021.
B. Research Methods

1. Research Design

The research will be conducted by survey with purposive random

sampling technique. Samples came from hospitalized patients. Samples were
collected by nasopharyngeal swab. The samples are extracted in
Laboratorium Riset Terpadu of Jenderal Soedirman University. The positive
result with Cycle Thresholds (CT) value under 25 were sent to the LIPI
Cibinong to performed Whole Genome Sequencing (WGS) and deposited on

6
 Global Initiative on Sharing All Influenza Data (GISAID). The result will be
analyzed and present with PANGO.
2. Research Parameter

The research variables observed in the experiment are variants of

SARS-CoV-2. The parameter observed in the experiment is a new variant of
COVID-19 spreading in Purwokerto, Banyumas, Jawa Tengah.
3. Research Procedures

3.1 RNA Extraction

3.1.1 Lysis Buffer Portion
Lysis buffer solution made of the Viral Lysis Buffer
(AVL Buffer) and Proteinase-K. For 1 reaction (rxn), we need
600 µl of AVL Buffer and 20 µl of Proteinase-K and multiples
depending on the number of reactions. Lysis buffer for 1 reaction
is prepared in the tube based on the amount of the samples.
3.1.2 RNA Extraction Using Liferiver Reagent
Lysis Buffer with 620 µl volumes, consisting of 600 µl of
AVL Buffer and 20 µl of Proteinase-K, were poured in to a
centrifuge tube with 1,5 ml volumes and added 300 µl of samples
into the tube, vortex and spin down for 3 seconds. The solution
was thermomixed at a temperature of 56⁰ C for 15 minutes and
added 600 µl of alcohol absolute and repeat the vortex and spin
down for 3 seconds. Transfer the sample solution of 760 µl of
volumes into the spin column. The supernatant is eliminated and
the collection tube was placed. Repeat the transfer and remove the
supernatant 2 times, after that add 500 µl of Washing Buffer A
(already mixed with 17 ml of alcohol absolute) and centrifuge at a
temperature between 4-80C, speed of 12.000 rpm for 1 minute.
Eliminate the supernatant, adds 500 µl of Washing Buffer B
(already mixed with 48 ml of alcohol absolute), and centrifuges at
12.000 rpm and a temperature between 4-80C for 3 minutes. The
collection tubes were replaced with recovery tubes. To wash away
unbound proteins, 50 µl of Elution Buffer were added and left the
solution at room temperature for 5 minutes. To mix the solution,

7
 repeat centrifuged at 12.000 rpm and a temperature between 4-
80C for 1 minute and remove the spin column. The tubes were
labeled to differentiate the samples.
3.1.3 Master Mix Configuration Using Liferiver Reagent

Calculate the volume of Master Mix and Enzyme Mix by

multiplying the volume of the reagent by the number of samples
to be tested, plus negative control, positive control, internal
control, and additional pipetting error volume. For 1 sample, we
need to perform 7 reactions with 133 µl of SuperMix and 7 µl of
Enzyme Mix. For the samples, we need to perform 9 reactions
with 171 µl of Super Mix and 9 µl of Enzyme Mix and spin down
to mix the solution.
3.1.4 RNA Amplification
Prepared the wells for PCR microtubes for samples, PC,
NC, and IC. RNA samples were taken as much as 5 µl and
transferred to a special PCR micro tubes provided which already
contained 19 µl of Super Mix and 1µl of Enzyme Mix. The RNA
samples were vortexes and spin down for 3 seconds. The prepared
plate is brought to the RT PCR device for examination with the
following settings:
Table 3.1 Settings for PCR Examination
45°C for 10 minutes 1 cycle Selections of Fluorescence
Channels
95°C for 10 minutes 1 cycle FAM Target
Gene
95°C for 15 seconds, 45 HEX/IC/JOE Target
60°C for 1 minute cycles Gene
Fluorescence measure Cal Red IC
in 60°C 610/ROX/TE
XAS RED
The positive result with Cycle Threshold (CT) value
under 25 were send to LIPI Cibinong to be analyzed using Whole
Genome Sequences (WGS).
3.2 Whole Genome Sequencing using Amplicon sequencing protocol
for SARS-CoV-2 v3 (LoCost)
3.2.1 cDNA Preparation

8
 The samples were prepared plus one negative control of
nuclease-free water. The frozen samples were vortexing and pulse
spin to collect the liquid. The components listed below were
mixed in the PCR strip tubes or plate by pipetting and spinning
down the tube using a vortex.
Table 3.2 Components for cDNA Preparation
N Component Volume
o
1 LunaScript RT SuperMix (5X) 2 µL
2 Template RNA 8 µL
Total 10 µL
The reactions were incubated with following requirement:
Table 3.3 Requirement for Incubation
Temperature Time
25 °C 00:02:00
55 °C 00:10:00
95 °C 00:01:00
Hold at 4 °C
3.2.2 Multiplex PCR
Q5 Hot Start High-Fidelity 2X Master Mix was
prepared by setting up the two PCR reactions per sample as
follows in strip-tubes or plates by pipetting and pulse spin the
tube to collect the tube with the following components:
Table 3.4 Component for Q5 Hot Start High-Fidelity 2X Master Mix

Component Reaction 1 Reaction 2

5X Q5 Reaction Buffer 5 µL 5 µL
10 mM dNTPs 0.5 µL 0.5 µL
Q5 Hot Start DNA
0.25 µL 0.25 µL
Polymerase
V3 Pool 1 (10µM) 4 µL 0 µL
V3 Pool 2 (10µM) 0 µL 4 µL
Nuclease-free water 12.75 µL 12.75 µL

9
 After the Q5 Hot Start High-Fidelity 2X Master Mix
was made, 2.5 µL volumes of cDNA were added to each PCR
reaction, gently mixed by pipetting and pulse spin the tube to
collect liquid at the bottom of the tube. To multiplex the Q5 Hot
Start High-Fidelity 2X Master Mix, the reactions were thermomix
using thermal cycler with the following program:
Table 3.5 Thermal Cycler Program for Thermomix The
Reaction
Step Temperature Time Cycles
Heat Activation 98°C 00:00:30 1
Denaturation 98°C 00:00:15 25-35
Annealing 65°C 00:05:00 25-35
Hold 4°C Indefinite 1
III.2.3. PCR Dilution
The PCR post-clean up concentration were performed by
tagging the strip-tubes/plate and combining the following
volumes of each PCR reaction for 10 µL each sample:
Table 3.6 Component for PCR Dilution
Component Volume
Pool 1 PCR reaction 2.5 µL
Pool 2 PCR reaction 2.5 µL
Nuclease-free water 45 µL
Total 50 µL

III.2.4.Native Barcoding
The amplicon pools were barcoded by using a one-pot
native barcoding approach. A new PCR strip-tube /plate was set
up to prepare a new reaction for each sample with the following
components:
Table 3.7 Component for Master Mix of End-preparation
Component Volume
PCR dilution from previous step 3.3 µL
Ultra II End Prep Reaction Buffer 1.2 µL
Ultra II End Prep Enzyme Mix 0.5 µL
Nuclease-free water 5 µL

After the preparation of the Master Mix finish, incubate

the reaction at room temperature for 15 minutes, at the

10
temperature of 65°C for another 15 minutes, and incubate on ice
for 1 minute. New PCR strip-tube /plate was set with the
following reaction for each sample:
Table 3.8 Component for Native Barcoding Reaction
Component Volume
End-preparation reaction mixture 0.75 µL
NBXX barcode 1.25 µL
Blunt/TA Ligase Master Mix 5 µL
Nuclease-free water 3 µL
Total 10 µL

The reactions were incubated three times, incubated at

room temperature for 20 minutes, at a temperature of 65°C for
another 10 minutes, and incubated on ice for 1 minute. The
reactions are pooled in the new 1,5 mL Eppendorf tube pool, all
one-pot barcoding reactions together. After that, 0.4x volume of
SPRI beads was added to the sample tube and mixed gently. SPRI
with 0.4x of volume is sufficient to bind 400 bp amplicons in the
presence of ligation buffer. Mix again the reaction by vortexing
and pulse centrifuge to collect all liquid at the bottom of the tube.
Incubate for 5 minutes at room temperature. Place on a magnetic
rack and incubate for 2 minutes or until the beads have pelleted
and the supernatant is completely clear. Carefully remove and
discard the supernatant, careful not to touch the bead pellet. The
SFB with 250 µL of volumes was added, and pipette mixing
resuspended beads thoroughly. Pulse centrifuge to collect all
liquid at the bottom of the tube and place on the magnet. Remove
supernatant and discard. Repeat the steps to perform a second
SFB wash, then pulse centrifuge and remove any residual SFB.
200 µL volume of room-temperature 70 % volume ethanol to
bathe the pellet were added. Carefully remove and discard
ethanol, careful not to touch the bead pellet. Pulse centrifuge to
collect all liquid at the bottom of the tube and carefully remove as
much residual ethanol as possible using a P10 pipette; with the
tube lid open, incubate for 1 minute or until the pellet loses its

11
shine. Resuspend pellet in 30 µL 10 Milimolar (mM) Tris pH 8.0,
mix gently by either flicking or pipetting, and incubate for 2
minutes. Place on magnet and transfer sample to a clean 1.5 mL
Eppendorf tube, ensuring no beads are transferred into this tube.
Quantify 1 µL volume of the barcoded amplicons using
the Quantus Fluorometer using the ONE dsDNA assay.
Concentration will vary depending on the number and Ct of
samples. Lambda DNA standard with 400 ng/µL volume was
removed from the freezer and left on the ice to thaw. Remove
ONE dsDNA dye solution from the fridge and allow it to come to
room temperature. Set up two 0.5 mL tubes for calibration, label
them 'Blank' and 'Standard,' then add 200 µL ONE dsDNA Dye
solution to each tube. The Lambda DNA standard 400 ng/µL
standard is mixed by pipetting then adding 1 µL to one of the
standard tubes. Mix each sample vigorously by vortexing for 5
minutes and pulse centrifuge to collect the liquid, then incubate at
room temperature for 2 minutes before proceeding. Selection
'Calibrate' then 'ONE DNA' then place the blank sample in the
reader then select 'Read Blank.' Now place the standard in the
reader and select 'Read Std.' The required number of 0.5 mL tubes
were set up for the number of DNA samples to be quantified and
labeled the tubes on the lids to avoid marking the sides of the tube
as this could interfere with the sample reading.
The ONE dsDNA dye solution with 199 µL volume
was added to each tube, and 1 µL of each user sample was added
to the appropriate tube. After that, mix each sample vigorously by
vortexing for 5 minutes and pulse centrifuge to collect the liquid.
Allow all tubes to incubate at room temperature for 2 minutes
before proceeding. On the Home screen of the Quantus
Fluorometer, select `Protocol, ` then select `ONE DNA` as the
assay type. On the home screen, navigate to 'Sample Volume' and
set it to 1 µL, then 'Units' and set it to ng/µL. Load the first
sample into the reader and close the lid. The sample concentration
is automatically read when the lid closes and repeated for all the

12
 samples. The value displayed on the screen is the dsDNA
concentration in ng/µL were recorded.

III.2.5.MinION Sequencing
The flowcell is primed, and a 15 ng volume of sequencing
library was loaded onto the flowcell. Prepare Sequencing buffer
(SQB), Loading beads (LB), Flush buffer (FLB), and Flush tether
(FLT) were thaw at room temperature before placing on ice. The
30 µL volume of FLT was added to the FLB tube and mixed well
by vortexing. Suppose required to place a new MinION flowcell
onto the MinION by flipping open the lip, pushing one end of the
flowcell under the clip, and pushing down gently. The inlet port
cover is rotated clockwise by 90° so that the priming port is
visible. Take a P1000 pipette and tip and set the volume to 800
µL. Place the tip in the inlet port and hold perpendicularly to the
flowcell plane. Remove any air from the inlet port by turning the
volume dial anti-clockwise. The FLB (plus FLT) with 800 µL
volume loaded into the flow cell via the inlet port dispense slowly
and smoothly, trying to avoid introducing any air bubbles, rest for
5 minutes, and gently lifts the SpotON cover to open the SpotON
port. Another 200 µL volume of FLB (plus FLT) is loaded into
the flow cell via the inlet port, and this will initiate a siphon at the
SpotON port to allow you to load the library dilution. The new
tubes were used to prepare for sequencing the library dilution
with the following components (Table 3.8.). The library dilution
reaction was mixed gently and added the library dilution with 75
µL volume to the flow cell via the SpotON sample port in a
dropwise fashion. The SpotON sample port cover was replaced,
insert the bug into the SpotON port and closed the inlet port, then
closed the MinION lid.
Table 3. 9 Component for Sequencing of Library Dilution
Component Volume
SQB 37.5 µL
LB 25.5 µL
Library 12 µL

13
 Total 75 µL

The sequencing run started using MinKNOW. Before

that, plug the MinION into the computer and wait for the MinION
and flowcell to be detected. Select flow cell 'FLO-MIN106', select
the flowcell, and click the 'New Experiment' button from the
drop-down menu. On the New experiment popup screen, select
the running parameters. Name the run in the experiment field that
leaves the sample field blank. Then click 'Kit' then 'Selection,'
select LSK109 as there is no option for native barcoding
(NBD104). In the 'Run Options' button, set the run length to 6
hours. If the run once sufficient data has been collected as
determined using RAMPART, stop. Click 'Base-calling,' select
'fast base calling. The outcome is the number of files MinKNOW
will write to a single folder. By default, this is set to 4000 but can
be reduced to make RAMPART update more frequently. To
begin, click 'Start run.' The run progress is monitored using the
MinKNOW interface. The result uploaded in the GISAID.

3.3 Pangolin COVID-19 Lineage Assigner

Pangolin COVID-19 Lineage Assigner can be accessed in
Pangolin Website Application, https://pangolin.cog-uk.io/. The COVID-
19 sequences from the samples are assigned by uploading the samples
sequences in the ‘Drop Zone’ (Figure 3.1). Click “Select a fasta file to
upload” and select a (multi)fasta file of sequences, then start the analysis
by clicking on the ‘Start Analysis’ button (Figure 3.2). The sequences
will be uploaded, and the webserver will start analysis (Figure 3.2).

14
Figure 3.1 Pangolin COVID-19 Lineage Assigner ‘Drop Zone’

Figure 3.2 Pangolin COVID-19 Lineage Assigner ‘Start Analysis’

On culmination of the lineage assignment, a measure of the
possibility of the assignment will be displayed in the column marked
'Assignment probability,' the probability of that sequence being the
assigned lineage vs. any other lineage (Figure 3.3). Extra information
regarding the lineage to which the sample has been assigned can be
obtained by clicking the tick button next to the sample (Figure 3.3).

Figure 3.3 Pangolin COVID-19 Lineage Assigner ‘Result Review’

15
 To visualize the geographical and temporal distribution,
Microreact is used. Next to each lineage assignment are 2 icons (Figure
3.4). Clicking on the globe icon will open an interactive Microreact
visualization that shows the lineage selected in the context of global
SARS-CoV-2 samples (Figure 3.4). The results can be downloaded in
CSV format.

Figure 3.4 Visualized the geographical and temporal distribution in

Microreact
4. Data Analysis
The research data is obtained from the samples analyzed with WGS
and uploaded in GISAID. The analysis we used employed the Pango
classification system, which facilitates the classification and nomenclature of
SARS-CoV-2 genetic lineages, containing molecular signatures that can be
helpful to track its introduction/emergence and spread.

16
C. Research Framework

Respondents have symptoms of COVID-19

Nasopharynx swab was performed to be collect the samples

↓
Samples were extracted and proceed using RT-PCR, positive result
with CT < 25 were collected

↓
Whole Genome Sequencing (WGS) were performed using Amplicon
sequencing protocol for SARS-CoV-2 v3 (LoCost)

↓
The result uploaded in GISAID

↓
Data Analysis :
Genetic lineage assignment was undertaken using the Pango
system.

↓
Result

↓
Conclusion

17
 OBSERVATION FORM

Table 3. 10 Samples Extraction Result

Sample No.
No Date Code Laboratorium Result ORF1b RdRp IC
1
2
3
Etc.

Table 3. 11 Proceeds Samples for Whole-Genome Sequencing

No Lineage Amount Percentage (%)

Etc.

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 IV. RESEARCH SCHEDULE

The research entitled "Detection of New Variants COVID-19 from Purwokerto, Banyumas, Central Java " will be held with schedule as listed in
table 4.1.
Table 4. 1. Research Schedule
Months
No Activity March '21 April '21 July '21 Feb '22 March '22 April '22 June '22 July '22
1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4
1. Samples Collection
2. Sending Samples to Laboratory
3. Samples Extraction and
Sequencing Result
4. Outline Preparation
5. Preparation of Research Proposals
6. Research Proposal Seminar
7. Data Analysis
8. Thesis Draft Preparation
8. Research Result Seminar

19
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