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Finding SNP Sites in the Whole Genome of a Plant Using GATK

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As I have discussed in the previous blog, we already have aligned SAM (.sam) file to be further utilized for analyzing genomic variations. In this post, I am going to instruct the steps involved in the analysis of genomic variations among the closely related organisms. In particular, I am presenting the workflow pipeline for the GATK (Genome Analysis Tool Kit) HaplotypeCaller for detecting SNP (Single Nucleotide Polymorphism) sites and their annotation by snpEff .  Note: In order to carry out SNP sites detection by GATK, there should be SAM file of the whole genome sequence of an organism, preinstalled GATK software version  4.1.0.1  and above  (McKenna et al., 2010)  with its accessory packages, Samtools, and snpEff   4.3t   (Cingolani et al., 2012)  in Linux server. Image 1. a screenshot of a home page of gatk website (source: https://software.broadinstitute.org/gatk/) In brief 1. get the SAM file of the whole genome sequence of the sample organism; 2. do SNP
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How to Align Fastq Files of Sample Genomic DNA Sequence to Reference Genome

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Since the aim of this blog is to share the information on various aspects of plants and plant sciences, this time, I am blogging about the bioinformatics tools that help study about the genome of organisms including plants.    The application of  BWA  software or any other software that allows alignment of fastq (.fq) files of the sample genomic DNA sequence to  reference genome  is an essential step before carrying out further investigations such as the genome analysis. In this post, I am going to present the instruction for the alignment of quality trimmed fastq (.fq) files of a sample genome to the reference genome using BWA (Burrows-Wheeler Aligner) software. This instruction covers installing of BWA software, indexing the reference genome, quality trimming the raw fastq files, and aligning the quality trimmed  fastq  files to reference genome to get  SAM  (Sequence Alignment Map) file of a sample genome.  Image 1. The vines of Vitis vinifera cv. 'Pinot Noir' with be
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Some Fungi Degrade Polyurethanes

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Conidia and conidiophore of Cladosporium cladosporioides  (Source: commons.wikimedia.org) Fungi , a separate kingdom in living organisms, includes vast numbers of species with diverse usage to humanity and roles in the ecosystem. They are known to have a potential to reduce human-induced pollution by degrading numbers of toxic as well as non-toxic xenobiotics and transforming a wide variety of hazardous chemicals. In a recent study done by a group of scientists under the leadership of Ivano Brunner from Switzerland showed an ability of fungi to degrade plastic waste in the environment. The results are published in the PLOS ONE journal (Date: August 22, 2018) showing the power of fungi isolated from plastic debris floating in the shoreline of  Zurich lake, Switzerland, to degrade plastics.  Memory foam cushion made up of viscoelastic polyurethane (Source: commons.wikimedia.org) The team of researchers isolated over a hundred fungal strains and grouped them on the bas
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How to Determine Cytotoxic Activity of Crude extracts by MTT assay

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After total flavonoids content (TFC), total polyphenolics content (TPC), and antioxidant activity of different crude extracts of orchid including Dondrobium longicornu have been estimated, we decided to determine their cytotoxic activity using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay on human brain tumor cells ( U251 ) and cervical cancer cells ( HeLa ).  We employed previously established protocols (Mosmann, 1983; Sargent and Taylor, 1989) which is presented below point wisely.  Culture human brain tumor cells (U251) and cervical cancer cells (HeLa) in RPMI 1640 medium and incubate them under 5% CO 2 at 37 ° C for 48 hours to reach  80% confluence. Harvest by gentle scraping with a cell scraper and resuspended in the medium.  Dispense 100 μ L of such medium with 5 × 10 3 cells into each well of 96-well microtiter cell culture plate and incubated under the same conditions of cell culture for 48 hours to allow adherence and growth of cel
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The Rapid Method of Estimating Antioxidant Activity and Total Polyphenols Content of Sample Plant Extracts

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Estimation of total polyphenols content Figure1. The appearance of bluish-black colour after the addition of reagents on sample plant extracts during the estimation of total polyphenols content.  We estimated  total polyphenol content  by a standard protocol (Singleton et al . 1999 and Stankovic 2011) with slight modification. Briefly, we  separately mixed  each plant extract (0.5 ml; 1 mg/ml)  with the Folin-Ciocalteu phenol reagent (2.5 ml; 10%) and aqueous sodium bicarbonate solution (2.5 ml; 7.5%). Then, the reaction mixture was allowed to stand for about 45 minutes and the absorbance was measured at 765 nm using the UV- spectrophotometer (Thermo Fisher Scientific, Genesystem -10.5).The calibration curve was plotted using gallic acid as standard in ethanol (absolute) using the concentration series of 25, 50, 75, and 100 µg/ml. Here, the reagent mixture with the  same volume of ethanol instead of plant extract solution was used as a blank. On the basis of this calibration
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How we performed the estimation of total flavonoid content ?

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This time I am sharing a method for the estimation of total flavonoid content in our experiments. We Flowchart. the process of estimating total flavonoids content. followed a previously done standard protocol  (Quettier  et al.  2000 and Stankovic 2011)  with slight modification.  Firstly, we  mixed  e ach plant extract (2 ml; 0.5 mg/ml) separately  with aluminium chloride solution (1 ml; 2 %). Then, we allowed the reaction mixture to stand for an hour at room temperature. Again, we measured the absorbance of the mixture at 415 nm using the UV-spectrophotometer. We  obtained  calibration curve (see Fig 1.) using quercetin solution series (25, 50 75, and 100 µg/ml) as a standard. Here, we used the reagent mixture with the same volume of ethanol instead of plant extract solution as a blank. Thus, the obtained curve was calibrated to estimate the concentration of total flavonoid in each plant extract. The total flavonoids content was expressed in terms of the milligram of qu
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How did we perform preliminary screening for the phytochemical classes?

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While performing the laboratory tasks for my dissertation work, I searched many articles and books to have a reliable protocol which would be easy to follow. However, I found either very standard books which compiled the test methods in lengthy literature or research articles mentioning test procedures only briefly; easy to understand but not easy to follow. At last, I consulted protocols by three authors (which are named below) and did according to the state of our laboratory.  In this post, I am going to share the information about how we (me and my junior fellow) detected the presence of phytochemical classes in orchid sample   extract s. The aim of this post is  to provide reliable and easy to follow protocol for undergraduate and graduate students who wish to detect phytochemical classes  in sample plant extracts.   Basically, we followed protocols established by Habourne (1998), Trease and Evans (1983) with minor modifications according to the condition of our labora
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