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ABCD of Sarcoma Genomics - A Beginners Note Experience from The Yellow Ribbon

WHAT IS GENOMICS?

Genomics is the study of an organism’s/ individual genome (total genetic complement- sum of genes and intergenic sequences). The complete genome study is required to:

- Determine what is missing from the genome

- Identify genes and pathways that are difficult to study biochemically.

- Study evolution of the organism and genome.

- To understand and find every gene that is in the pathway of interest.

- Identify variant sequences that may have subtle phenotypes.

The genomic study is done by the use of high throughput DNA (Deoxyribonucleic acid) sequencing and bioinformatics to assemble and analyze the function and structure of entire genomes. Researchers can use the information obtained to search for genetic variations and/ or mutations that may play a role in the development or progression of a disease.


WORKFLOW:

Genomic study or research is not just the laboratory work, there is a lot of effort that goes into collecting, storing and transporting the samples (tissue) for the research work. The Yellow Ribbon is an orthopedic oncology organization with the mission to help every individual with bone or soft tissue tumor get the best outcome in the world by involving the best professionals and adapting cutting edge technology warranted, without any cost barrier. At TYR (“The Yellow Ribbon”), we collected fresh samples of tissues (A sample of biopsy while taking was dipped into RNA later) of a few sarcoma patients which were then stored in RNAlater. This can be obtained from any of the genomics laboratories. RNAlater is an aqueous, nontoxic tissue storage reagent that rapidly permeates tissue to stabilize and protect cellular RNA in situ in unfrozen specimens.


FFPE blocks of several sarcoma patients were also considered for this study. FFPE (Formalin-fixed paraffin-embedded) is a form of preservation and preparation for biopsy specimens that aids in examination, experimental research, and diagnostic/ drug development. A total of 39 samples of various patients diagnosed with Ewing Sarcoma, Osteosarcoma, Synovial sarcoma and Giant cell tumor were collected. For the FFPE samples, the corresponding slides were marked for the tumor rich region by the pathologist. The collected fresh samples and the FFPE blocks, slides were then sent to the 4baseCare Genomics laboratory for further processing. 4baseCare is an Illumina Accelerator Company, engaged in the development of cutting-edge precision oncology solutions, using advanced genomics and next-gen digital health technology, based in Bangalore, India to personalize patient care in oncology. 4baseCare Genomics in Research Collaboration with TYR further processed the samples.

The laboratory processing involves the high throughput sequencing and the bioinformatics part in the laboratory. The high throughput sequencing used in this case is NGS (Next Generation Sequencing) and the NGS platform used is Illumina. There are many other sequencing methods such as Sanger sequencing, Maxam- Gilbert sequencing, Shotgun sequencing which are the conventional methods. However, NGS is the most preferred method of sequencing over conventional methods although the basic principle behind Sanger sequencing and NGS are similar. The main advantages of NGS over classical Sanger sequencing are its speed, cost, sample size and accuracy.

NGS amplifies millions of copies of a particular fragment of DNA in a massively parallel fashion and the output data generated after the sequencing will be in the form of “reads”. These reads would be further analyzed by comparing with the information already available using the computational program. The whole process of NGS can be categorized into 4 steps:

a) Library preparation.

b) Bridge building and amplification.

c) DNA sequencing.

d) Data analysis.

In the pre-processing stage the DNA is first obtained from the tissue sample. Then the steps of NGS are carried out.

Library preparation:

The library preparation is the combination of two reactions, fragmentation and ligation. The fragmentation of cDNA or DNA fragments is done by restriction digestion i.e., a cutting process. After that, the smaller DNA fragments are ligated with the known DNA sequence also known as adapters. Once the adapters are ligated, the library of smaller DNA fragments is generated. The unbound DNA fragments are then washed by the washing buffer (Phosphate Buffered Saline).

The DNA fragments are checked for its quantity and quality using capillary electrophoresis and bioanalyzers.

The first column is the DNA ladder which is used as a reference to measure the size of the DNA fragments. The good quality DNA fragments size should be in the range of 300-400bp (base pairs). If it is greater or lesser than this range, then the quality of the sample may not be good or there may be any technical errors which have occurred during the library preparation.

Graphs of the ladder and sample are obtained from the bioanalyzers.

Once the quality of the sample is assured to be good, the further steps are carried out.


Bridge building and amplification:

The short oligonucleotide sequences are immobilized on the solid surface which is complementary to the adapter sequences. Once the library of the fragmented DNA is loaded into the flow cell, it is bound with immobilized oligos on the solid surface by bridge amplification. In bridge amplification, the DNA fragments bend over and bind to the next oligonucleotide which creates a bridge. A primer binds to this DNA sequence and is amplified vertically. The cluster of the DNA sequence is generated.


Sequencing:

The sequencing method is based on Sequencing By Synthesis (SBS). DNA polymerase, connector primers and 4 dNTP with base specific fluorescent markers were added to the reaction system. Only one base is added at a time during the sequencing process. At the end of the synthesis process, all the unused free dNTP and DNA polymerase are eluted. A fluorescence signal is excited by a laser which is recorded by optical equipment. The optical signal is then converted into a sequencing base by computer analysis. This generates multiple sequencing data bases for the DNA sequence.


Data Analysis:

NGS generates a very large quantity of data which cannot be manually analyzed and hence uses Bioinformatics for the data analysis. The read generated by the sequencing can be aligned to a reference genome/ normal genome. By doing this we can identify any addition, deletion or variation in the sequence.


GENOMICS IN SARCOMAS

The tumor mutational burden and microsatellite instability information obtained from NGS data have potential as biomarkers for response to immunotherapy in certain sarcoma subtypes including angiosarcoma. Identification of hallmarks associated with 'BRCAness' and homologous recombination repair defects in leiomyosarcomas and osteosarcomas may predict sensitivity to poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors. Lastly, the use of NGS for evaluating cancer predisposition in sarcomas may be useful for early detection, screening and surveillance. Currently, the implementation of NGS for every sarcoma patient is not practical or useful. However, adopting NGS as a complementary approach in sarcomas with complex genomics and those with limited treatment options has the potential to deliver precision medicine to a subgroup of patients, with novel therapies such as immune checkpoint and PARP inhibitors.


SUMMARY

All in all, this genomic study for sarcomas is going to give an insight into the type and extent of variations of genes responsible for a particular type of sarcoma. It also helps in understanding the functions of genes. This study has the potential to play a pivotal role in advancement of Gene therapy for sarcomas in the future.


Interested to know more about the NGS platform, Illumina?

Then, do come back to check our upcoming blog about it.

Comentarios


ABCD of Sarcoma Genomics - A Beginners Note Experience from The Yellow Ribbon

WHAT IS GENOMICS?

Genomics is the study of an organism’s/ individual genome (total genetic complement- sum of genes and intergenic sequences). The complete genome study is required to:

- Determine what is missing from the genome

- Identify genes and pathways that are difficult to study biochemically.

- Study evolution of the organism and genome.

- To understand and find every gene that is in the pathway of interest.

- Identify variant sequences that may have subtle phenotypes.

The genomic study is done by the use of high throughput DNA (Deoxyribonucleic acid) sequencing and bioinformatics to assemble and analyze the function and structure of entire genomes. Researchers can use the information obtained to search for genetic variations and/ or mutations that may play a role in the development or progression of a disease.


WORKFLOW:

Genomic study or research is not just the laboratory work, there is a lot of effort that goes into collecting, storing and transporting the samples (tissue) for the research work. The Yellow Ribbon is an orthopedic oncology organization with the mission to help every individual with bone or soft tissue tumor get the best outcome in the world by involving the best professionals and adapting cutting edge technology warranted, without any cost barrier. At TYR (“The Yellow Ribbon”), we collected fresh samples of tissues (A sample of biopsy while taking was dipped into RNA later) of a few sarcoma patients which were then stored in RNAlater. This can be obtained from any of the genomics laboratories. RNAlater is an aqueous, nontoxic tissue storage reagent that rapidly permeates tissue to stabilize and protect cellular RNA in situ in unfrozen specimens.


FFPE blocks of several sarcoma patients were also considered for this study. FFPE (Formalin-fixed paraffin-embedded) is a form of preservation and preparation for biopsy specimens that aids in examination, experimental research, and diagnostic/ drug development. A total of 39 samples of various patients diagnosed with Ewing Sarcoma, Osteosarcoma, Synovial sarcoma and Giant cell tumor were collected. For the FFPE samples, the corresponding slides were marked for the tumor rich region by the pathologist. The collected fresh samples and the FFPE blocks, slides were then sent to the 4baseCare Genomics laboratory for further processing. 4baseCare is an Illumina Accelerator Company, engaged in the development of cutting-edge precision oncology solutions, using advanced genomics and next-gen digital health technology, based in Bangalore, India to personalize patient care in oncology. 4baseCare Genomics in Research Collaboration with TYR further processed the samples.

The laboratory processing involves the high throughput sequencing and the bioinformatics part in the laboratory. The high throughput sequencing used in this case is NGS (Next Generation Sequencing) and the NGS platform used is Illumina. There are many other sequencing methods such as Sanger sequencing, Maxam- Gilbert sequencing, Shotgun sequencing which are the conventional methods. However, NGS is the most preferred method of sequencing over conventional methods although the basic principle behind Sanger sequencing and NGS are similar. The main advantages of NGS over classical Sanger sequencing are its speed, cost, sample size and accuracy.

NGS amplifies millions of copies of a particular fragment of DNA in a massively parallel fashion and the output data generated after the sequencing will be in the form of “reads”. These reads would be further analyzed by comparing with the information already available using the computational program. The whole process of NGS can be categorized into 4 steps:

a) Library preparation.

b) Bridge building and amplification.

c) DNA sequencing.

d) Data analysis.

In the pre-processing stage the DNA is first obtained from the tissue sample. Then the steps of NGS are carried out.

Library preparation:

The library preparation is the combination of two reactions, fragmentation and ligation. The fragmentation of cDNA or DNA fragments is done by restriction digestion i.e., a cutting process. After that, the smaller DNA fragments are ligated with the known DNA sequence also known as adapters. Once the adapters are ligated, the library of smaller DNA fragments is generated. The unbound DNA fragments are then washed by the washing buffer (Phosphate Buffered Saline).

The DNA fragments are checked for its quantity and quality using capillary electrophoresis and bioanalyzers.

The first column is the DNA ladder which is used as a reference to measure the size of the DNA fragments. The good quality DNA fragments size should be in the range of 300-400bp (base pairs). If it is greater or lesser than this range, then the quality of the sample may not be good or there may be any technical errors which have occurred during the library preparation.

Graphs of the ladder and sample are obtained from the bioanalyzers.

Once the quality of the sample is assured to be good, the further steps are carried out.


Bridge building and amplification:

The short oligonucleotide sequences are immobilized on the solid surface which is complementary to the adapter sequences. Once the library of the fragmented DNA is loaded into the flow cell, it is bound with immobilized oligos on the solid surface by bridge amplification. In bridge amplification, the DNA fragments bend over and bind to the next oligonucleotide which creates a bridge. A primer binds to this DNA sequence and is amplified vertically. The cluster of the DNA sequence is generated.


Sequencing:

The sequencing method is based on Sequencing By Synthesis (SBS). DNA polymerase, connector primers and 4 dNTP with base specific fluorescent markers were added to the reaction system. Only one base is added at a time during the sequencing process. At the end of the synthesis process, all the unused free dNTP and DNA polymerase are eluted. A fluorescence signal is excited by a laser which is recorded by optical equipment. The optical signal is then converted into a sequencing base by computer analysis. This generates multiple sequencing data bases for the DNA sequence.


Data Analysis:

NGS generates a very large quantity of data which cannot be manually analyzed and hence uses Bioinformatics for the data analysis. The read generated by the sequencing can be aligned to a reference genome/ normal genome. By doing this we can identify any addition, deletion or variation in the sequence.


GENOMICS IN SARCOMAS

The tumor mutational burden and microsatellite instability information obtained from NGS data have potential as biomarkers for response to immunotherapy in certain sarcoma subtypes including angiosarcoma. Identification of hallmarks associated with 'BRCAness' and homologous recombination repair defects in leiomyosarcomas and osteosarcomas may predict sensitivity to poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitors. Lastly, the use of NGS for evaluating cancer predisposition in sarcomas may be useful for early detection, screening and surveillance. Currently, the implementation of NGS for every sarcoma patient is not practical or useful. However, adopting NGS as a complementary approach in sarcomas with complex genomics and those with limited treatment options has the potential to deliver precision medicine to a subgroup of patients, with novel therapies such as immune checkpoint and PARP inhibitors.


SUMMARY

All in all, this genomic study for sarcomas is going to give an insight into the type and extent of variations of genes responsible for a particular type of sarcoma. It also helps in understanding the functions of genes. This study has the potential to play a pivotal role in advancement of Gene therapy for sarcomas in the future.


Interested to know more about the NGS platform, Illumina?

Then, do come back to check our upcoming blog about it.

Comentarios


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