An overview of the methodology used in “Penaeus monodon gene discovery project: The generation of an EST collection and establishment of a database” Essay

STB 3062 – Functional Genomics

Assignment 01:

Select one published paper in Expressed Sequence Tags and depict briefly the methodological analysis involved in fixing an EST database. Explain how this method can be used to detect new cistron. Highlight the paper you have selected in the mentions subdivision.

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Group 1

Student name

:

Lim Wei Yong

Matric figure

:

36741

Lector

:

Associate Professor Hairul Azman Roslan

Assignment due day of the month

:

08 April 2015

The chief diary used is entitled “Penaeus Monodoncistron find undertaking: The coevals of an EST aggregation and constitution of a database” with the mention doi:10.1016/j.gene.2006.07.012

Background of the survey

Shrimps refer to one of the decapod crustaceans that are consumed world-wide and hence it has high economic value in the seafood market. ThePenaeus Monodonor locally known as black tiger shrimp is one of the major seafood green goods in Asia ( Rosenberry, 2004 ) . However, due to two major jobs, the agriculture forP. Monodonhas declined late and replaced by another type of runtLitopenaeus vannamei. The first job addressed is the trouble of using confined genteelness toP. Monodon. Besides that,P. Monodonhas ever been susceptive to shrimp pathogens such as white topographic point syndrome and xanthous caput viruses ( Tassanakajon et al. , 2006 ) . With the purpose to cultivate the runt with disease-resistant and fast turning traits, it is of import to to the full understand the genome information. However, the shrimp biological cognition peculiarly on the reproduction, control of growing and immune system is non good understood. Hence, a high-density familial linkage map ofP. Monodonis perfectly required for physical function and preliminary genomic surveies.

One manner of analyzing the genome ofP. Monodonis by EST analysis. Although there are several genomic surveies on EST analysis have been carried out for the penaeidae runts, scientists merely manage to qualify little figure of cistrons out of the big genome of about 2.5 Gbp and sedimentation into the EST database ( Wilson et al. , 2002 ) . In order to increase the figure of shrimp cistrons in the public database, a large-scale complementary DNA sequencing work will be done to supply of import genomic resource for theP. Monodontiger runt. In the survey conducted by Tassanakajon et Al. ( 2006 ) , there will be a entire sum of 10,100 high quality EST ringers generated from 15 complementary DNA libraries resourced from assorted tissues ofP. Monodonthat is reared under normal every bit good as stress status. Examples of the tissues that are used include haemocyte, eyestalk, hepatopancrease, lymphoid organ, ovary, and haeatopoietic tissue ( Tassanakajon et al. , 2006 ) . In add-on to building of EST database forP. Monodon, EST database besides allowed the designation of cistron and discovery new cistron with unknown maps in theP. Monodonevery bit good as several tissue-specific transcripts that have impact on the immune response.

Methodology

Experimental animate beings

Harmonizing to Tassanakajon et Al. ( 2006 ) , the experimental runts chosen were juvenile runt ( 2-4 months ) sized 15-25 g for building of complementary DNA libraries from different tissues as mentioned above except for ovarian and eyestalk. Ovary and eyestalk tissues were taken from big female runt ( Eyestalk tissues resulted in two libraries: one for 0-1stphase of ovary development, followed by 3rd-4Thursdayphase ovary development phase ) . Other than that, stressed juvenileP. Monodonwas besides prepared from injection of each inoculant of white topographic point syndrome virus ( WSSV ) , xanthous caput virus ( YHV ) , andVibrio harveyibacteriums. There was besides a library created from heat-stressedP. Monodonwithout any injections. Haemolymph of 10 persons treated with WSSV are extracted and pooled for entire RNA extraction. Lymphoid variety meats of 12 juveniles injected with YHV ; haemocytes and lymphoid organ of theV. harveyiinoculated runt were besides pooled for entire RNA extraction. Meanwhile, the haemocyte pellet from heat stressed juvenile at 35 °C for 1 hr was besides collected.

Standard and Normalized complementary DNA libraries

In the survey conducted by Tassanakajon et Al. ( 2006 ) stated that the building of complementary DNA libraries started off with the usage of TRI-REAGENT to pull out the full RNA from each tissues type. Then, messenger RNAs were purified and five mcgs of it was subjected to cDNA synthesis utilizing the Uni-Zap XR complementary DNA synthesis kit. Those cDNAs with size more than 500 bp were selected and ligated onto the dephosphorylated EcoR1/Xho1 digested Uni-Zap vector and packaged in vitro. The libraries were amplified and so converted to pBluescript libraries by in vivo deletion. About 1000 ringers were indiscriminately selected from each library and sent for nucleotide sequencing.

Normalized complementary DNA libraries were constructed for lymphoid organ and hepatopancrease from their standard complementary DNA libraries in order to efficaciously stamp down the complementary DNA library size for extremely abundant transcripts ( Tassanakajon et al. , 2006 ) .

Deoxyribonucleic acid Sequencing and EST Assembly

Next, the Deoxyribonucleic acid sequencing was done by utilizing MegaBace1000 DNA Analysis System whereby the plasmid DNA was sequenced by dideoxy-chain expiration sequencing reaction ( Tassanakajon et al. , 2006 ) . Each complementary DNA insert was sequenced from the 5’ terminal with automatically remotion of bad and unwanted vector sequence. Harmonizing to Takasuga et Al. ( 2001 ) , 5’ ESTs were utile as it contained the cryptography sequence. After that, analysis of sequence was done via the comparing of sequenced informations against the GenBank database utilizing BLASTX plan. The low quality sequences for illustration those sequences with length less than 100 bp are removed by utilizing SeqClean ( Tassanakajon et al. , 2006 ) . In add-on to that, TIGR Gene Indices Clustering Tools ( TGICL ) had been used to constellate and piece the sequences.

EST Database Design and Implementation

The database of EST was designed and implemented by MySQL database system. A BLAST agent written in “Perl” programming linguistic communication was used to pass on with the BLAST waiter and store the consequences into the database system ( Tassanakajon et al. , 2006 ) . In other words, the EST database created was used to hive away the EST sequence informations and those consequences obtained from the BLAST hunts. This enabled a creative activity of ESTs database specifically used by the runtP. Monodon.The database user interfere besides allowed users to entree, hunt, blast, and modify their sequences easy and efficaciously.

Gene Discovery

Harmonizing to the consequences published by the Tassanakajon et Al. ( 2006 ) in the diary, there were 10,261 ringers indiscriminately selected from 15 complementary DNA libraries ( 13 criterion libraries and 2 normalized libraries ) have been sequenced to bring forth ESTs. However, 161 low quality ESTs were discarded and a sum of 10,100 high quality ESTs were analysed by utilizing the TIGR Gene Indices Clustering Tools ( TGICL ) . After the bunch and collection, the ESTs were categorized into 917 overlapping contigs and 3928 singletons which resulted in a sum of 4845 putative transcripts. Each of these non-redundant ESTs was so subjected to similarity hunts against the sequences available in public EST database such as GenBank utilizing the BLASTX plan with an e-value cut off & lt ; 10-4. Consequences showed that 51.2 % out of the sum blasted 4845 putative transcripts can non fit with the database. The other 48.8 % of the transcripts showed important homology to known cistrons. These unmatch ESTs could likely go the fresh cistrons that have non been found before. For farther cistron find for the speciesP. Monodon, as shortly as the genome information of other beings, particularly those closely related species become available, the figure of shrimp cistrons will besides be increasing and therefore more new cistrons would be identified and discovered.

In the survey itself, the writers besides manage to detect several cistrons response to microbic and heat emphasis challenge. From the EST analysis of haemocyte complementary DNA libraries showed that the ESTs encoding for ribosomal proteins had significantly increased in theV. harveyiand WSSV treated libraries as compared to the healthy 1. Putative cistrons figure had besides increased evidently in the microbic challenged libraries. However, there was no important addition of ESTs encoding for heat aghast proteins. Furthermore, several interesting cistrons were identified from the eyestalk complementary DNA libraries. There were a few tissue-specific complementary DNA found such as the molt-inhibiting endocrine ( MIH ) and pigment scattering endocrine ( PDH ) resulted from the BLAST hunt ( Tassanakajon et al. , 2006 ) . Besides that, haemocyanin molecule that usually found in arthropods and mollusk is besides expressed in theP. Monodonhepatopancrease with fungicidal and antiviral effects ( Tassanakajon et al. , 2006 ) . In add-on to that, cistrons encode for the alpha-NAC protein and dystrobrevin-like protein were besides identified in the hematopoietic tissue whereby these two proteins are involved in the blood cell distinction ( Tassanakajon et al. , 2006 ) .

Main Mention

Tassanakajon, A. , Klinbunga, S. , Paunglarp, N. , Rimphanitchayakit, V. , Udomkit, A. , Jitrapakdee, S. , … Lursinsap, C. ( 2006 ) .Penaeus Monodoncistron find undertaking: The coevals of an EST aggregation and constitution of a database.Gene 384, 104-112.

Supporting Mentions

Rosenberry, B. ( 2004 ) .World runt agriculture 2004. San Diego, CA: Shrimp News International.

Takasuga, A. , Hirotsune, S. , Jitohzono, A. , Suzuki, H. , Aso, H. , & A ; Sugimoto, T. ( 2001 ) . Constitution of a high throughput EST sequencing system utilizing poly ( A ) tail-removed complementary DNA libraries and finding of 36,000 bovine ESTs.Nucleic Acids Res. 29( 22 ) , 108.

Wilson, K. , Li, T. , Whan, V. , Lehnert, S. , Byrne, K. , Moore, S. , …Benzie, J. ( 2002 ) . Familial function of the black tiger runtPenaeus Monodonwith amplified fragment length polymorphism.Aquaculture,204( 3-4 ) , 297–309.