Genome technology & applications
partim FK
NEXT GENERATION SEQUENCING
THE IMPACT OF NGS TECHNOLOGIES ON HUMAN GENETICS
Genetic disorders are frequent but many are not identified. This could be done with NGS.
WHOLE EXOME SEQUENCING: WES
If there is an abnormality present in the genome of all patients with a specific genetic disorder, we
could identify the causative gene (= whole genome sequencing, WGS). BUT we don’t need the whole
genome, we could just sequence the exome. Only 1-2% of the genes in the genome is coding. Most
identified diseases are caused by aberrations in the coding sequences. WES is more affordable to
discover novel disease genes.
To go from a genome sequencing to an exome sequencing, you need enrichment of the whole
exome. You take the genomic DNA fragments and ligate adapters and take probes with a biotin-label.
These probes encode all the coding sequences. If you then use magnetic beads, you take away all
probes bound to coding sequences and you can get rid of the non-coding sequences.
You can also do it on an array instead of in solution. The coding probes are now attached to the array
and the DNA fragments are added. When you wash, only the coding sequences are kept.
If you don’t filter, you get a huge amount of variants that could be recessive. So you need a selection
strategy. You want to get rid of the ‘normal variation’. Then you are left with a couple or even one
variation. The software is very important: you need to use the right filters so you don’t filter away too
many or too little variants. Next you do Sanger Sequencing in other patients to confirm the
hypothesis.
, TRIO APPROACH
For identifying novel, as yet unknown genetic disorders.
If a child has intellectual disability, without it being seen in the parents, it
could be a de novo mutation. You could sequence the child and both
parents and compare the sequences. If you find an abnormality in the
parent and the child then it is not likely to be disease-causing, but if it is
present in the child but not in the present this could be suggestive of it
being disease-causing.
Large cohorts of patients have been sequenced, and there are quite a few genes in which you only
find one or two mutations in 5000 patients. If you do larger cohorts you can exclude these genes
because they aren’t likely to be disease-causing, since most patients don’t have it.
MULTIPLEX TARGETED SEQUENCING (MIP)
If you want to screen 10-20K patients for a selective, well-defined set of genes, you could use MIPs.
You design probes for a large series of genes you’re interested in, and you hybridize them and ligate
the probes to each other. You can then capture the target and sequence it. It is cheap and efficient if
you want to screen thousands of patients.
LONG READ SEQUENCING
For short reads, you typically use Illumina or BGI technology because they’re fast and relatively
cheap.
LONG READ SEQUENCING: CGG REPEAT EXPANSION IN FRAGILE X SYNDROME
Fragile X syndrome is caused by a CGG repeat expansion in front of a gene. if you have more than
200 repeats it gets methylated and the gene won’t be functional anymore (no more transcription).
This results in the disease.
This is diagnosed now by Southern Blotting or other methods. You would like the whole sequence of
the repeat so they are all pretty laborious. You can’t really use PacBio or Illumina because the reads
can be 1000 bp long, which means you have 300-400 repeats. If you use Illumina, reads are typically
150 bp: not able to estimate the length and PacBio is too expensive but it can be used to detect the
expansion! Nanopore is lacking a little behind but still also used to show an expansion in the reads.
partim FK
NEXT GENERATION SEQUENCING
THE IMPACT OF NGS TECHNOLOGIES ON HUMAN GENETICS
Genetic disorders are frequent but many are not identified. This could be done with NGS.
WHOLE EXOME SEQUENCING: WES
If there is an abnormality present in the genome of all patients with a specific genetic disorder, we
could identify the causative gene (= whole genome sequencing, WGS). BUT we don’t need the whole
genome, we could just sequence the exome. Only 1-2% of the genes in the genome is coding. Most
identified diseases are caused by aberrations in the coding sequences. WES is more affordable to
discover novel disease genes.
To go from a genome sequencing to an exome sequencing, you need enrichment of the whole
exome. You take the genomic DNA fragments and ligate adapters and take probes with a biotin-label.
These probes encode all the coding sequences. If you then use magnetic beads, you take away all
probes bound to coding sequences and you can get rid of the non-coding sequences.
You can also do it on an array instead of in solution. The coding probes are now attached to the array
and the DNA fragments are added. When you wash, only the coding sequences are kept.
If you don’t filter, you get a huge amount of variants that could be recessive. So you need a selection
strategy. You want to get rid of the ‘normal variation’. Then you are left with a couple or even one
variation. The software is very important: you need to use the right filters so you don’t filter away too
many or too little variants. Next you do Sanger Sequencing in other patients to confirm the
hypothesis.
, TRIO APPROACH
For identifying novel, as yet unknown genetic disorders.
If a child has intellectual disability, without it being seen in the parents, it
could be a de novo mutation. You could sequence the child and both
parents and compare the sequences. If you find an abnormality in the
parent and the child then it is not likely to be disease-causing, but if it is
present in the child but not in the present this could be suggestive of it
being disease-causing.
Large cohorts of patients have been sequenced, and there are quite a few genes in which you only
find one or two mutations in 5000 patients. If you do larger cohorts you can exclude these genes
because they aren’t likely to be disease-causing, since most patients don’t have it.
MULTIPLEX TARGETED SEQUENCING (MIP)
If you want to screen 10-20K patients for a selective, well-defined set of genes, you could use MIPs.
You design probes for a large series of genes you’re interested in, and you hybridize them and ligate
the probes to each other. You can then capture the target and sequence it. It is cheap and efficient if
you want to screen thousands of patients.
LONG READ SEQUENCING
For short reads, you typically use Illumina or BGI technology because they’re fast and relatively
cheap.
LONG READ SEQUENCING: CGG REPEAT EXPANSION IN FRAGILE X SYNDROME
Fragile X syndrome is caused by a CGG repeat expansion in front of a gene. if you have more than
200 repeats it gets methylated and the gene won’t be functional anymore (no more transcription).
This results in the disease.
This is diagnosed now by Southern Blotting or other methods. You would like the whole sequence of
the repeat so they are all pretty laborious. You can’t really use PacBio or Illumina because the reads
can be 1000 bp long, which means you have 300-400 repeats. If you use Illumina, reads are typically
150 bp: not able to estimate the length and PacBio is too expensive but it can be used to detect the
expansion! Nanopore is lacking a little behind but still also used to show an expansion in the reads.
