TITLE: Mutational Spectra of SARS-CoV-2 orf1ab polyprotein and Signature mutations in the United States of America
AUTHORS: Shuvam Banerjee, Sohan Seal, Riju Dey, Kousik Kr. Mondal, Pritha Bhattacharjee

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Pandemic COVID-19 outbreak has been caused due to SARS-COV2 pathogen, resulting millions of infection and death worldwide, USA being on top at the present moment. The long, complex orf1ab polyproteins of SARS-COV2 play an important role in viral RNA synthesis. To assess the impact of mutations in this important domain, we analyzed 1134 complete protein sequences of orf1ab polyprotein from NCBI Virus database from affected patients across various states of USA from December 2019 to 25</span><span style = "vertical-align:super;">th</span><span>April, 2020. Multiple sequence alignment using Clustal Omega followed by statistical significance was calculated. Four significant mutations T265I (nsp 2), P4715L (nsp 12) and P5828L and Y5865C (both at nsp 13) were identified in important non-structural proteins, which function either as replicase or helicase. A comparative analysis shows 265T>I, 5828P>L and 5865Y>C are unique to USA and not reported from Europe or Asia; while one, 4715P>L is predominant in both Europe and USA. Mutational changes in amino acids are predicted to alter structure and function of corresponding proteins, thereby it is imperative to consider the mutational spectra while designing new antiviral therapeutics targeting viral orf1ab.</span></div></div>

[STEPS]
?. [ORF1ab protein sequence retrieval from the database]
Retrieve the protein sequences through the NCBI Virus database.
Specific input was "SARS-CoV-2"
?. [ORF1ab protein sequence retrieval from the database]
Refine the output with a sequence length of 7100 from the initial 7050.
The length of the target orf1ab polyprotein is 7096.
?. [ORF1ab protein sequence retrieval from the database]
In total, 1307 sequences will be retrieved, among them 125 sequences were from Asia, 42 from Europe and 1134 solely from USA.
?. [Screening of submitted sequences and selection of study sample]
Eliminate all incomplete sequences or sequences with undermined residues (mentioned as X).
?. [Screening of submitted sequences and selection of study sample]
In total, 867 orf1ab polyprotein complete sequences deposited from 31 different states within the US were considered for the study (Group A).
All the above-mentioned sequences from Asia (Group B) and Europe (Group C) were also selected.
?. [Multiple Sequence Alignment (MSA) and analysis of mutational spectra]
Align multiple sequences of each of the above-mentioned groups using Clustal Omega.
?. [Multiple Sequence Alignment (MSA) and analysis of mutational spectra]
Subdivide sequences in Group A into 31 subgroups depending on the regional source from which the sequence was originated.
?. [Multiple Sequence Alignment (MSA) and analysis of mutational spectra]
MSA was conducted encompassing all subgroups using ancestral orf1ab polyprotein sequences of Wuhan (YP_009742608, comprising of 7096 residues) as reference sequence.
?. [Multiple Sequence Alignment (MSA) and analysis of mutational spectra]
Set gap opening penalty and gap extension penalty at 12 and 2, respectively, to ensure that unnecessary gaps are not created during alignment and alterations are visualised easily.
?. [Multiple Sequence Alignment (MSA) and analysis of mutational spectra]
Thoroughly screen alignment results to determine the exact locaitons of the mutations and alteration linked to that position.
?. [Calculation of statistical significance to detect signature mutations of USA]
Calculate the number of occurance of each mutated variant in Group A, B, and C.
?. [Calculation of statistical significance to detect signature mutations of USA]
Divide by the total number of sequence submitted under that group.
The proportion of each variant in Group A would be a/x and likewise for the other groups.
?. [Calculation of statistical significance to detect signature mutations of USA]
Caluclate the p-value through Z-score using the proportion of the mutant and sample size to establish whether the occurance of that mutant variant in USA (Group A) is significantly higher in comparison to Asia and Europe (Group B and C).
Two-tailed p values were calculated using 0.05 as the significance level. Thus attempt has been made to identify the signature mutations in the region of orf1ab polyprotein.
?. [Homology Modelling and simulation of protein structure]
Report structures of the associated non-structural proteins for wild type at I-Tasser server.
The structures were not available for the varied amino acid alteration.
?. [Homology Modelling and simulation of protein structure]
To identify the alteration, generate secondary structure by using the Homology Modelling method by I-Tasser.
?. [Homology Modelling and simulation of protein structure]
Superimpose the alteration with the wild type using UCSF Chimera and PyMOL for easy visualization and comparison.