Balamurugan

Antiquity and Peopling of the Deccan –
an Ancient DNA approach

Ref: 048/RUSA/MKU/2020-2021
BALAMURUGAN M
PROGRAMMER
Department of Genetics
School of Biological Sciences
Madurai Kamaraj University
Objectives
Objectives
Objective 1
S.No TITLE of Standard Operating Protocol(SOP)
SOP For NGS Analysis - Ancient Human, Animal And Microbial Data
1.Alignment Of Ancient Animal Sequenced Data Against The Corresponding Whole Animal Reference Genome Using BWA-MEM
2.Alignment Of Ancient Animal Sequenced Data Against Another Animal Sequenced Data Using BWA-MEM (In Case Where There
Is No References Sequences But Only The Submitted Sequenced Data In Fastq.Gz R1 & R2 Are Available In European Nucleotide
Archive-Enaeg.Kangayam Breed Of Cattle)
3.Alignment Of Ancient Animal Sequenced Data Against The Corresponding Whole Animal Reference Genome Using BWA-Aln
4.Use Of Parallel Codes To Align Multiple Samples Against A Single Reference Genome
5.Use Of VCFTOPOP To Generate Phylogenetic Tree With Vcf.Gz As Input Files
6.Use Of VEP (Variant Effect Predictor From Ensembl) For Comparison Of Variants Across 1K Project, GNOMAD Databases
I (Population Wise Variant Frequency Prediction)
7.Analysis Of Large Galaxy Output Filesusing ‘Awk’ And ‘Grep’ Commands
8.Human Analysis (Alignment Of Human Sequenced Data Against The Whole Human Reference Genome , Similarly Alignment
Against The Mitochondrial Reference Genome, Haplogroup Assignment, Extracting Mitochondrial Reads From An Aligned Bam
File)
9.PCA(For Human Samples)
10.Metagenome Analysis : A)Taxonomic Classification By KRAKEN2, BRAKEN, B)Alignment Of Metagenome Against The Bacterial
Reference Genome
11.Installation Of Tools In The Server For Ancient Genomic Analysis
12.Transfer Of Data Via FTP (Raw Data Transferred To Broad Institute FTP Server)
SOP For Transcriptome Analysis
13.Analysis Of Alternative Splice Variants From Microarray Using TAC
II 14.Analysis Of Alternative Splice Variants From RNA sequencing Using AltAnalyze
15.Analysis Of Mutation From Transcriptome
16.MicroRNA Exploration
17.Gene Set Enrichment Analysis Using Enrichr
18.Other Alignment Tools Used

SOP For NGS Analysis-Bacteria
19.Identification Of Bacterial Species
III 20.Bracken Analysis
21.Metaphlan2 Analysis
22.Analysis Of Damage Frequency For The Bacterial Species

SOP For NGS Analysis - Ancient Human Genomes
23.Separating Ancient Damaged Reads using PMD tools
IV 24.Mitochondrial Haplogroup assignment
25.Global Blast Result Analysis
26.Parallel Codes To Align multiple samples against Rice
SOP For Human Cancer Genomics
27.Gene Set Enrichment Analysis (Gsea)
V 28.Single-Sample Gene Set Enrichment Analysis (Ssgsea)
29.Deepcc
30.Nmf Consensus Clustering
31.Z Score-Based Pathway Activation Prediction
32.Scripts For Pathway Action Prediction Analysis

SOP For Human Whole Genome Analysis
33.Quality Check using FASTQC
34.Trimming And Merging using SeqPrep
35.Mapping To Human Reference Genome using BWA
36.Sorting using Samtools
37.Remove Duplicates using Samtools
38.Mapping Quality using Samtools
39.Damage Profiler for screening ancient DNA damages
40.Preprocessing For Vcf File Generation using
VI 41.Tabix
42.Bcf Variant Calling
43.Bcf To Vcf Conversion
44.Statistics From Vcf File
45.Annotation Of Vcf File With Dbsnp Database
46.Vcf To Bed File Using Plink
47.Snp File Conversion Using Eigenstrat
48.Merging Of Huge Population
49.Principal Component Analysis

Completed SOPs
• I.1 Alignment Of Ancient Animal Sequenced Data Against The Corresponding Whole Animal Reference Genome Using
BWA-MEM
Input : Fastq files
Output : Damage Plot
• I.2.Alignment of ancient animal sequenced data against another animal sequenced data using BWA-MEM
Input : Fastq files
Output : Damage Plot
• II.1 Analysis of alternative splice variants from Microarray Using TAC
Input : (50 Tumor and 15 Normal) CELL FILES from Affymetrix array
Output : Alternative splicing results for input samples.,Differential gene expression, Plots and graphs for
expression profile(Analysis_1.tacx).
• II.2 Analysis Of Alternative Splice Variants From Rna sequencing Using AltAnalyze
Input : Fastq files
Output : Alternative splicing results for input samples.
Differential gene expression.
Sashimi Plots and graphs for expression profile.
Objective 2
Problem 1 : To Develop a Shell Script for Piping multiple commands in ancient DNA data analysis
1.Alignment of Sequenced data against Whole Reference Genome
2. Mitochondrial Haplogrep Analysis
Advantages:
• User friendly
• Avoid repetition in typing the commands
• Reduce time
Problem 2 : To Develop a pipeline for Alignment Set Extraction using Python Script
• Input : Readid file (.xlsx file) and whole aligned reads file (.txt)
• Output : Extract the reads from the whole aligned reads file (.txt) based on the readid in the Readid file (.xlsx file)
Advantages:
• User friendly
• Reduce time
Python script to extract a subset

from a large datasets
Problem 3 : To Develop a pipeline for Meta Merge using Python Script
• Input : Gene Expression data (.csv file)
• Output : Merged Gene Expression data (.csv file)
Gene Expression data(.csv) files
Extracting Common Genes from all the Expression

Cohort to Merge into single one
Merged Gene Expression data(.csv) files
Advantages:
• Pipeline can run in any device
• Avoid Local CPU Usage
Problem 4 : To Develop a pipeline for Removal of duplicate variants from Ensembl VEP using Python Script
• Input : (.txt) file that contains 50 columns with duplicates
• Output : (.txt) file removed duplicates rows based on column 20 (Variants)
Advantages:
• Pipeline can run in any device
• Reduce Running time
Objective 3
Using AWS for the RNA & DNA analysis

• Samples of (30 out of 30)RNA Sequence samples and Samples of (14 out of 14) EXOME Sequence samples are
uploaded successfully in AWS.
• RNA Analysis Nextflow Pipeline Execution for Differential Gene Expression Analysis – (Local)
Objective 4
Objective 5
Software Installation in Server

Installed Softwares
•Fastqc
•Damage Profiler
•Seqtk
•Parallel
•Bcftools
•bgzip
•Tabix
•Ry_compute
•Pmdtools
•Haplogrep
•Vcf_utils
•Qualimap
•Careal
Objective 6,7 & 8
Planned Publication
 Balamurugan Mahalingam, Anisha Marina , Kumaresan Ganesan, Simplified and User friendly Pipelines for Ancient
Genomic data Analysis
 Anisha Marina, Dheerkha Darsha Kannan, Kumaresan Ratha, Ramesh Masethung, Ajay Kumar Ramamoorthy,
Balamurugan Mahalingam, Sivanantham N Ramalingam, Rajan K, and Kumaresan Ganesan, Genomic evidence spotting
cattle, Bos species, in the archaeological habitation site Keeladi, Tamil Nadu, India.
 Kumaresan Ganesan, Anisha Marina , Gayathri Venugopalan, Balamurugan Mahalingam Laboratory Manual for
Ancient Genome Data Analysis
 Raja Rajeswari Sethuraman , Balamurugan Mahalingam, Thangamari Vengatesh Balamurugan,Kannan Subramanium,

Kumaresan Ganesan, A Comparative Analysis of Cancer Transcriptomes using different classification methods

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Antiquity and Peopling of the Deccan –

an Ancient DNA approach

S.No TITLE of Standard Operating Protocol(SOP)

5.Use Of VCFTOPOP To Generate Phylogenetic Tree With Vcf.Gz As Input Files

7.Analysis Of Large Galaxy Output Filesusing ‘Awk’ And ‘Grep’ Commands

9.PCA(For Human Samples)

11.Installation Of Tools In The Server For Ancient Genomic Analysis

13.Analysis Of Alternative Splice Variants From Microarray Using TAC

II 14.Analysis Of Alternative Splice Variants From RNA sequencing Using AltAnalyze

15.Analysis Of Mutation From Transcriptome

17.Gene Set Enrichment Analysis Using Enrichr

18.Other Alignment Tools Used

19.Identification Of Bacterial Species

III 20.Bracken Analysis

22.Analysis Of Damage Frequency For The Bacterial Species

23.Separating Ancient Damaged Reads using PMD tools

IV 24.Mitochondrial Haplogroup assignment

25.Global Blast Result Analysis

26.Parallel Codes To Align multiple samples against Rice

SOP For Human Cancer Genomics

27.Gene Set Enrichment Analysis (Gsea)

V 28.Single-Sample Gene Set Enrichment Analysis (Ssgsea)

30.Nmf Consensus Clustering

31.Z Score-Based Pathway Activation Prediction

32.Scripts For Pathway Action Prediction Analysis

33.Quality Check using FASTQC

34.Trimming And Merging using SeqPrep

35.Mapping To Human Reference Genome using BWA

36.Sorting using Samtools

37.Remove Duplicates using Samtools

38.Mapping Quality using Samtools

39.Damage Profiler for screening ancient DNA damages

40.Preprocessing For Vcf File Generation using

42.Bcf Variant Calling

43.Bcf To Vcf Conversion

44.Statistics From Vcf File

45.Annotation Of Vcf File With Dbsnp Database

46.Vcf To Bed File Using Plink

47.Snp File Conversion Using Eigenstrat

48.Merging Of Huge Population

49.Principal Component Analysis

1.Alignment of Sequenced data against Whole Reference Genome

2. Mitochondrial Haplogrep Analysis

Python script to extract a subset

Gene Expression data(.csv) files

Extracting Common Genes from all the Expression

Merged Gene Expression data(.csv) files

Using AWS for the RNA & DNA analysis

Software Installation in Server

 Raja Rajeswari Sethuraman , Balamurugan Mahalingam, Thangamari Vengatesh Balamurugan,Kannan Subramanium,

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