What is Genome Assembly?

Genome assembly involves piecing together short DNA sequence reads generated by sequencing platforms (e.g., Illumina, PacBio, Oxford Nanopore) into longer, contiguous sequences called contigs. This can be performed as:

• De Novo Assembly: Without a reference genome.
• Reference-Guided Assembly: Using a reference genome to guide the assembly process.

Step 1: Preparing Your Data

Before starting the assembly, ensure that your raw sequencing data is high quality.

1. Input Data

   • Short Reads: Illumina sequencing generates short, accurate reads ideal for scaffolding.
   • Long Reads: PacBio and Nanopore sequencing provide long reads for resolving repetitive regions.

2. Quality Control (QC)
   Use tools like FastQC or MultiQC to assess the quality of your reads:

   fastqc reads.fastq multiqc .

   Look for issues like low-quality bases, adapter contamination, or overrepresented sequences.

3. Read Trimming and Filtering
   Trim low-quality bases and adapters using Trimmomatic or Cutadapt:

   trimmomatic PE reads_R1.fastq reads_R2.fastq trimmed_R1.fastq trimmed_R2.fastq \ ILLUMINACLIP:adapters.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:36

Step 2: Choosing an Assembly Strategy

Select an assembly strategy based on your data type:

• Short-Read Assemblers:

  • SPAdes: Popular for microbial genomes.
  • Velvet: Fast for smaller genomes.

• Long-Read Assemblers:

  • Canu: Ideal for long-read datasets.
  • Flye: Versatile for small and large genomes.

• Hybrid Assemblers:

  • MaSuRCA: Combines short and long reads.
  • Unicycler: Optimized for bacterial genomes.

Step 3: Running the Assembly

3.1. SPAdes (Short-Read Assembly)

SPAdes is an excellent choice for small genomes, such as bacteria.

The output includes assembled contigs (contigs.fasta) and scaffolds (scaffolds.fasta).

3.2. Canu (Long-Read Assembly)

Canu is designed for high-error long reads from PacBio or Nanopore.

The output will be in canu_output/genome.contigs.fasta.

3.3. Hybrid Assembly with Unicycler

Unicycler combines short and long reads for improved assemblies.

Step 4: Assessing Assembly Quality

After assembly, evaluate its quality using the following tools:

1. QUAST
   QUAST generates assembly statistics, such as N50, genome size, and GC content:

   quast contigs.fasta -o quast_output

2. BUSCO
   BUSCO checks genome completeness by identifying conserved genes:

   busco -i contigs.fasta -o busco_output -l fungi_odb10 -m genome

3. Assembly Graph Visualization
   Visualize assembly graphs with Bandage:

   Bandage load assembly_graph.gfa

Step 5: Post-Assembly Steps

1. Polishing
   Improve assembly accuracy using tools like Pilon (for short reads) or Racon (for long reads).

   racon long_reads.fasta mapped_reads.sam contigs.fasta > polished_contigs.fasta

2. Scaffolding
   Link contigs into scaffolds using tools like SSPACE or Opera-LG if required.

3. Annotation
   Annotate the assembled genome using Prokka for prokaryotes or Maker for eukaryotes.

   prokka --outdir annotation_output --prefix genome contigs.fasta

Step 6: Sharing and Archiving

1. Submit to Public Repositories
   Share your assembly in databases like NCBI GenBank, ENA, or DDBJ.

2. Metadata Preparation
   Include detailed metadata for your submission, such as organism name, sequencing platform, and coverage.

Best Practices

• Always perform quality checks at each stage to ensure data integrity.
• Use multiple tools to cross-validate results when working with complex genomes.
• Document parameters and software versions for reproducibility.

Conclusion

Genome assembly is a powerful process that transforms raw sequencing data into a coherent representation of an organism’s genome. By following this step-by-step guide, you can successfully assemble genomes and uncover valuable biological insights. Whether you’re assembling a microbial genome or tackling the complexities of a eukaryotic genome, these tools and strategies will set you on the path to success.

Project Description: Projects involves high throughput analysis of data mostly generated by massively parallel sequencing (RNA-Seq and small-RNA-Seq), microarrays and related platforms. We are looking for highly motivated and bright individuals interested in high-throughput cutting-edge data analyses methods in genomics (computational positions). Available positions: Applications are invited from suitable candidates in both, the Max Planck India Partner Program and the CRP Wheat Program for openings at the levels:

Post Name-Qualification-Salary:
Project assistant – Master’s – Rs. 14000
Project fellow (junior data analyst) – Masters + research experience – Rs. 16000
Research fellow (senior data analyst) – Masters + adequate research experience/desirable skill sets – Rs. 22000
Research Associated – PhD (< 1yr) /> 1 yr experience – Rs. 28000 / Rs. 32000
Essential qualification: MSc/MTech/PhD (or other suitable qualification) in discipline related to bioinformatics, computational biology, computer application (or equivalent)/ ‘Advance Post-Graduate Diploma in Bioinformatics’. Proficiency in one of the programming languages or statistics (proficient in R for example) is compulsory.
Desirable qualification: 1. Programming experiences in at least one low level language such as C/C++ and one scripting language such as Perl/Python/PHP and knowledge of SQL/MySQL. 2. Substantial experience in the linux or other unix environments. 3. Experience of working in projects on Bioinformatics, Genetics or Biological application areas/Computational and Statistical analysis (e.g. using R or Matlab). Experience in the field of genomics (NGS, microarrays, genome annotation), database development and management, software development, systems and network biology (or related fields) will be preferred.
SELECTION PROCEDURE FOR INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH (IISER KOLKATA) – RESEARCH ASSOCIATE & MORE VACANCIES POST:

Candidates can apply on or before 30/04/2015
No Detailed information about the selection process is mentioned in the recruitment notification
HOW TO APPLY FOR RESEARCH ASSOCIATE & MORE VACANCIES IN INDIAN INSTITUTE OF SCIENCE EDUCATION AND RESEARCH (IISER KOLKATA):

Applications should contain CV along with brief description (maximum 1 page) of research conducted (highlighting skills and experience) till now. Applications should be sent by email to Shree P. Pandey, Department of Biological Sciences, IISER-Kolkata, Mohanpur Campus, West Bengal within 2 weeks. Interviews will be scheduled within 10 days of closing of applications. E-mail: sppiiserkol@gmail.com, sppandey@iiserkol.ac.in
For more details visit: http://www.iiserkol.ac.in/~sppandey

Key Findings:

1. Chromosomal Fusion and Its Molecular Signature:

   • The fusion site is located at 2q13–2q14.1 and is characterized by degenerate telomeric sequences appearing interstitially, indicating the historical head-to-head joining of ancestral chromosomes.
   • Despite being a signature of a past fusion event, these telomeric repeats are no longer functional and have undergone sequence degradation over time.

2. Extensive Duplications in the Surrounding Genomic Region:

   • The study identifies large-scale segmental duplications flanking the fusion site, with several of these regions duplicated and scattered across multiple chromosomes.
   • These duplications are predominantly located in subtelomeric and pericentromeric regions, suggesting their role in genomic instability and chromosomal evolution.

3. Paralogous Regions and Their Evolutionary Relationships:

   • A 168-kilobase (kb) segment near the fusion site has 98%–99% sequence identity with three regions on chromosome 9 (9pter, 9p11.2, and 9q13).
   • Another 67-kb region distal to the fusion site shows a high degree of homology to sequences in chromosome 22qter.
   • Additionally, a 100-kb segment exhibits 96% sequence identity with a region in chromosome 2q11.2.

4. Comparative Genomics and Evolutionary Implications:

   • By comparing the duplicated sequences and their arrangement in primates, the researchers traced the order of duplication events leading to their present distribution.
   • The presence of specific repetitive elements within these duplicated segments serves as evolutionary markers that help infer their historical rearrangements.
   • Some of these duplicated regions are associated with chromosomal inversion breakpoints, potentially contributing to evolutionary changes in primates.
   • Recurrent structural rearrangements in these regions have been linked to human chromosomal disorders.

Conclusions and Implications:

• The findings provide valuable insights into the structural evolution of human chromosome 2, which played a crucial role in human speciation.
• Understanding these segmental duplications and their evolutionary trajectories sheds light on genomic instability, which may contribute to human genetic diseases.
• The study highlights how large-scale chromosomal rearrangements, such as fusion and duplication, have influenced the evolutionary divergence of humans from other primates.

This research advances our understanding of human genome evolution and offers a foundation for studying the effects of structural variants in genetic disorders.

How To Apply: Eligible candidates required to report for the Interview at 9:00 A.M. sharp on 11-05-2015 (For Position Code No. 001 to 009) and 12-05-2015 (For Position Code No. 010 to 016). Candidates reporting after 10:00 A.M will not be allowed to attend the interview. Eligible candidates may appear before the Selection Committee for interview on the date and time mentioned above at CDRI, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow-226031. Eligible candidates must bring with them duly filled up application form (which can be downloaded from our website www.cdriindia.org), along with Original certificates as well as attested copies of certificates of examinations starting from matriculation, date of birth, caste certificate (in case of SC/ST/OBC) experience certificate, publication, if any and recent passport size photograph etc. Original documents are essential for verification of the particulars quoted by the candidate in the application form and candidate failed to produce original documents at the time of verification, shall not be allowed to attend the interview. Any request for relaxation in this regard shall not be entertained.
Detail of Interview: 11-05-2015
Age Limit: 28 Years

Installation

General Usage Guide

Data Preprocessing Guide

Specific Tool Guides:

• BBDuk
• BBMap
• BBMask
• BBMerge
• BBNorm
• CalcUniqueness
• Clumpify
• Dedupe
• Reformat
• Repair
• Seal
• Split Nextera
• Statistics
• Tadpole
• Taxonomy

https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/

Address of the bookmark: https://jgi.doe.gov/data-and-tools/bbtools/bb-tools-user-guide/

Employer: Jawaharlal Nehru University
Address: Dr. S. Gourinath, Principal Investigator, School Of Life Sciences, Jawaharlal Nehru University, New Delhi-110067
Email: not mentioned / provided for this job post
URL: http://www.jnu.ac.in/Career/currentjobs.htm
Phone: 011 2674 2575
Skills: not mentioned / required for this job post
Experience: Experience in molecular biology, structural biology and bioinformatics is desired
Education: M.Sc. in any field of life sciences.
Job Location: New Delhi, Delhi, India (View Jobs in New Delhi, Jobs in Delhi, Jobs in India)

Job Description: School of Life Sciences, Jawaharlal Nehru University vacancy of JRF / SRF / RA in CSIR funded Projec

Name of the Post: JRF / SRF / RA

Salary: As per rules

Required Job Profile:

Candidate must possess M.Sc. in any field of life sciences.

Desired Job Profile:

Candidate having NET - CSIR or UGC and experience in molecular biology, structural biology and bioinformatics is desired and experience with publication is preferred.

How to apply:

Eligible and interested candidates should need to apply with complete details to the above mentioned address on or before 08.05.2015.

Refer to http://www.jnu.ac.in/Career/currentjobs.htm

http://funprogramming.org/

Research Fellows Systems Biology job vacancy in C.R.Rao Advanced Institute of Mathematics, Statistics and Computer Science (AIMSCS)

JRF : Qualification - M. Sc in Bioinformatics, Systems Biology, M. Sc statistics, or M. Tech in Bioinformatics,

Pay Scale : Rs. 25,000

SRF : Qualification- Qualification prescribed for JRF with 2 years of research experience.

Pay Scale : Rs. 28,000*

No.of Post: 2

Desirable: Candidates should have strong background in Computational biology, bioinformatics, statistics and algorithmic development. In addition to that previous experience of working on Linux, bio-informatics, NGS data analysis and Basic knowledge of biology is desirable. Programming on any one of the programming languages (C, C++, perl, python) and statistical framework (e.g. R, matlab, etc.) is highly desirable.

More at http://www.crraoaimscs.org/jrf_application_form_2015.pdf

• -e Makes the line of code be executed instead of a script
• -n Forces your line to be called in a loop. Allows you to take lines from the diamond operator (or stdin)
• -p Forces your line to be called in a loop. Prints $_ at the end

• This counts the number of quotation marks in each line and prints it

  perl -ne '$cnt = tr/"//;print "$cnt\n"' inputFileName.txt

• Adds string to each line, followed by tab

  perl -pe 's/(.*)/string\t$1/' inFile > outFile

• Append a new line to each line

  perl -pe 's//\n/' all.sent.classOnly > all.sent.classOnly.sep

• Replace all occurrences of pattern1 (e.g. [0-9]) with pattern2

  perl -p -i.bak -w -e 's/pattern1/pattern2/g' inputFile

• Go through file and only print words that do not have any uppercase letters.

  perl -ne 'print unless m/[A-Z]/' allWords.txt > allWordsOnlyLowercase.txt

• Go through file, split line at each space and print words one per line.

  perl -ne 'print join("\n", split(/ /,$_));print("\n")' someText.txt > wordsPerLine.txt

• or in other words, delete every character that is not a letter, white space or line end (replace with nothing)

  perl -pne 's/[^a-zA-Z\s]*//g' text_withSpecial.txt > text_lettersOnly.txt

• perl -pne 'tr/[A-Z]/[a-z]/' textWithUpperCase.txt > textwithoutuppercase.txt;

• Print only the second column of the data when using tabular as a separator

  perl -ne '@F = split("\t", $_); print "$F[1]";' columnFileWithTabs.txt > justSecondColumn.txt

• One-Liner: Sort lines by their length

  perl -e 'print sort {length $a <=> length $b} <>' textFile

• One-Liner: Print second column, unless it contains a number

  perl">perl -lane 'print $F[1] unless $F[1] =~ m/[0-9]/' wordCounts.txt

You can also add your favourite NGS educational material, or workshop tutorial by commenting on this bookmarks for user benefit. 

Understanding the basics of genome sequencing:

Tutorial by Luke Jostins.

http://www.genetic-inference.co.uk/blog/2009/04/basics-sequencing-dna-part-1/

http://www.genetic-inference.co.uk/blog/2009/08/basics-sequencing-dna-part-2/

A window into third-generation sequencing

http://hmg.oxfordjournals.org/content/19/R2/R227.full.pdf

==============================================

NGS data analysis pipelines

• Detecting and annotating genetic variations using the HugeSeq pipeline  DOI: 10.1038/nbt.2134
• NARWHAL, a primary analysis pipeline for NGS data http://bioinformatics.oxfordjournals.org/cgi/content/abstract/28/2/284?etoc
• RseqFlow: Workflows for RNA-Seq data analysis  DOI: 10.1093/bioinformatics/btr441
• ngs_backbone: a pipeline for read cleaning, mapping and SNP calling using Next Generation Sequence  10.1186/1471-2164-12-285
• A framework for variation discovery and genotyping using next-generation DNA sequencing data  PubMed: 21478889
• SNiPlay: a web-based tool for detection, management and analysis of SNPs. Application to grapevine diversity projects  DOI: 10.1186/1471-2105-12-134 Abstract: http://www.biomedcentral.com/1471-2105/12/134/abstract
• WEP: a high-performance analysis pipeline for whole-exome data http://www.biomedcentral.com/1471-2105/14/S7/S11
• DDBJ read annotation pipeline: a cloud computing-based pipeline for high-throughput analysis of next-generation sequencing data. http://www.ncbi.nlm.nih.gov/pubmed/23657089
• GATK: a Toolkit for Genome Analysis http://www.broadinstitute.org/gatk/
• Metagenomics:http://www.nbic.nl/education/nbic-phd-school/course-schedule/ngsmetagenomics/
• RNASeq:http://www.nbic.nl/education/nbic-phd-school/course-schedule/ngsrnaseq/
• Bioinformatics and Seq courses: http://www.isb-sib.ch/training/training-activities-schedule/archive-2013.html
• Variant Detection (Model organism) Advanced tutorial https://docs.google.com/document/pub?id=1CuKkKylVDb03tnN7RSWl5EUzleetn0ctjmvaidPKLxM
• Variant Detection Introductory tutorial https://docs.google.com/document/pub?id=1ZRzrjjOCvtAu3m-IKL-rbJ1f4On60dDL_IEwG7oejdI
• Microbial de novo Assembly for Illumina Data Introductory tutorial https://docs.google.com/document/pub?id=1N3AB9ptISUu4zULqe1kXpVF0BDyGb5f5yzxWSJd_WNM
• RNAseq Differential Gene Expression Introductory tutorial https://docs.google.com/document/pub?id=1KbTiBHtvHLfPRZ39AY3uriazrINA8TJzgjjwn1zPP7Y

" Please add your favourite NGS link below in comment section for the benefit of bioinformatics community ". 

Address of the bookmark: http://chagall.med.cornell.edu/NGScourse/