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.

We have developed panHiTE, a comprehensive and accurate pipeline for TE detection in large-scale population genomes. It has been successfully applied to hundreds of plant population genomes, demonstrating its effectiveness and scalability.

For detailed instructions, please refer to the panHiTE tutorial.

Address of the bookmark: https://github.com/CSU-KangHu/HiTE

The next-generation sequencing included whole genome sequencing(WGS), transcriptome sequencing (whole cDNA sequencing, RNA-seq), digital gene expression sequencing (Tag-Seq), ChIP-Seq, and so on. And there are many sequencing platform to generate sequece, as well know Sanger/ABi(the frist generation), Solexa/illumina, SOLiD/ABi, 454/Roche. But thier sequence format is different, also they have different error type. High quality data is very important for further analysis or data mining. There are many pipeline for raw sequence quality analysis and control with few of process for reporting reads quality statistical details, trimming, filtering, and error correction. Please bookmarks them for the benefits of bioinformatics community.

https://code.google.com/p/biowiki/

https://code.google.com/p/ngs-pipeline/source/browse/#svn%2Ftrunk

NGSand Perl scripts https://code.google.com/hosting/search?q=NGS+perl&projectsearch=Search+projects

NGS and Python scripts https://code.google.com/hosting/search?q=NGS+Python&projectsearch=Search+projects

Address of the bookmark: https://code.google.com/hosting/search?q=bioinformatics&sa=Search

While your PhD or PostDoc application, it is more common that you got rejected by many professors. Don't disappoint reply it calmly.

In grad school, I shared a house with three Bioinformatics PhD students. One, when he applied to a particular professor, received a letter that said, essentially, "If you are applying because you want to enrich yourself, great. If you are applying because you want a job, you should know that you won't get one." I am trying to tell you this is because if you, with a good background in Bioinformatics, are passing up opportunities, you must be a strong candidate in many areas. Enrich yourself.

So, my suggestion is take a deep breath, forgot about all. Don’t take it personally. It's been usual processes while hunting for a good lab and professor. Take is positive, I am not sure why they reject, but don't worry perhaps the lab don't deserve you. Always remember there are billions of reasons not to hire someone for projects, especially in a research sector.

My suggestion, please do not whine about how you were a great research candidate for the post, and you just can't understand why they were so stupid as to have rejected you! This feeling will not win you any points in research, community. Especially, when in todays socially connected era everyone is linked. Remember, a nice E-mail saying, "I really wished to working with you on this project and I hope we cross paths again," is all you need to send to the professor. Send a thank you note to the professor. Thank them for the time they spend to judge you. In the future, If you and the professor (of your dream) are attending a bioinformatics conference, invite him/her to lunch (please remember to pay the bill). In today evolving scientific ere, always remember to build your solid network in order to get a job of interest. Join all possible networking sites like LinkedIn, ResearchGate, Acamedia, FB for the same reason. You as a researcher always build a bridge with student/researcher/colleague/professor who have the research potential to lead in research and hire you. Just because you didn't get this project, doesn't mean there isn't another that will open up in couple of month.

Mostly, jobs that are hard to get are hard to get. Only you can decide if the continued sacrifices are worth the expected payout. If it is, keep on plowing. Build relationships. Attend conferences.

Image ref @ JaSonYa

Webinar on 'An integrated RNA and DNA approach to unravel genetic regulation in cancer'

Abstract

Whole exome DNA sequencing (WES) or whole genome DNA sequencing (WGS) allows detection of mutations and polymorphisms in all exonic and genomic regions, respectively, while messenger RNA sequencing (RNA-Seq) enables quantitative analysis of gene expression. Mutations in the genome result in diverse transcriptional aberrations that can be missed in a stand-alone WES/WGS analysis. An integration of DNA variant analysis and RNA-Seq analysis enables one to investigate the consequences of genomic changes in the RNA transcripts including germline and somatic changes, imprinting, RNA editing and allele specific expression (ASE). In this webinar, we will demonstrate this integrated approach using Strand NGS to identify high confidence mutations, RNA editing events and ASE in cancer.

Webinar Details

Register here: http://www.strand-ngs.com/webinar_registration

About Speaker:

Dr. Veena Hedatale, has a PhD in Plant Genetics from The Radboud University, Netherlands focused on meiosis and recombination. Her prior academic experience at Cornell University was on genetic mapping and gene transformation in Rice. She has worked with Monsanto, and contributed to data mining, database development as well as gene/promoter/pathway discovery for traits related to yield and stress in crop species. At Strand, Veena has worked on Pharmacogenomic analysis of targets and Gene family analysis projects. Currently, she is part of the Strand NGS Application Science team and is involved in the analysis of next generation sequencing data.

Please feel free to contact us 24/5, for availing free online training or if you have any questions.

Abstract: Strand NGS supports an extensive workflow for the analysis and visualization of RNA-Seq data. The workflow includes Transcriptome / Genome alignment, Differential expression analysis with Statistical approach and Splicing events detection. Strand NGS also supports novel discovery like identification of novel genes, exons and Novel splice junctions, alongside it can also detect gene fusion events. Further downstream analysis such as GO and pathway analysis can be performed on the set of interesting genes. The product has an option to create pipelines for time consuming jobs which automates analysis and leaves more time for end data interpretation. This webinar will give an overview of the features in the RNA-Seq data analysis workflow in Strand NGS and also highlights on parameters within each feature that can be optimized depending on datasets and analysis needs.

Speaker: Mr. Sugandan Sivamani, Senior Application Scientist, Strand Life Sciences

Date: 9th Nov, Session 1 for SAPK/ APFO: 2:30 PM IST Date: 9th Nov, Session 2 for AFO/ EMEA: 9:00 AM PST

Register here http://www.strand-ngs.com/webinar_registration

Project Fellow (Biological Sciences) Pay Scale: Rs. 16,000/- + 30 % HRA per month
Educational Requirements: M.Sc./B.Tech in life sciences/Biological sciences with at least 55 % marks
Experience Requirements: Research experience.
Details will be available at: http://www.igib.res.in/sites/default/files/24July2015.pdf

No of Post: 01
How To Apply: 1. Please fill up the proforma by clicking on the following link HR Online Form. 2. Candidate cannot apply for more than two posts. Last date of receiving application is 12-07-2015. No application would be entertained with “result awaited” status or after due date. List of shortlisted candidates will be put up on CSIR-IGIB website. No TA/DA will be paid to the candidates to attend the interview. The engagement shall be as per guidelines of CSIR/Funding agency. Candidates will have an option to give reply in Hindi. Note: The shortlisted candidates, have to report at 09:00 AM at Mall Road Campus, Delhi – 110007 on the day of interview along with any Photo ID card, (without photo ID card interview will not be conducted). 3 copies of updated signed C. V. (clearly mentioning Date of Birth and Highest Qualification with percentage), Dissertation (if any), PhD thesis (if any) and original certificates/Self attested photocopies for verification.
Detail of Interview: 24 July, 2015 at 10:30 AM
Age Limit: 28 Years

1. Research Associate – 1 No.

Rs. 36,000/38,000/40,000 per month for I, II and III year + 20% HRA

Essential : Doctoral degree in relevant subject from recognized University/ Institutes

Desirable: Research experience in molecular biology and bioinformatics.

Interested candidates can send their complete CV in plain paper with a passport size photograph, with details of marks secured in all subjects from plus two stage (with proof, full postal address, sex, date of birth, community etc., along with additional qualification or experiences and two address of references whom could be contacted.

DEPARTMENT OF MICROBIOLOGY SCHOOL OF LIFE SCIENCES UNIVERSITY Dr. N. THAJUDDIN Professor & Head Dean, Faculty of Science, Technology & Engineering Tiruchirappalli – 620 024, India, Phone: +91 431 2407082; Mobile +91 098423 79719; E-mail: nthaju2002@yahoo.com

Application should reach the Principal Investigator on or before 5.8.2015 by Speed post/Couriers/Email (nthaju2002@yahoo.com), with subject printed as “Application for Research Associate /Technical Assistant /Lab attendant” in the envelop. Qualifying candidates will be short listed and communicated with date of interview. No TA and DA will be given for attending the interview. Address for Communication Dr.N.Thajuddin Principal Investigator Department of Microbiology Bharathidasan University Tiruchirappalli – 620 024, Tamil Nadu.

Advertisement: www.bdu.ac.in/adv/microbiology_advt.pdf