Limits...
Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing.

Eboreime J, Choi SK, Yoon SR, Arnheim N, Calabrese P - PLoS ONE (2016)

Bottom Line: These rates far exceed the well documented human genome average frequency per base pair (~10-8) suggesting a non-biological explanation for our data.By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles.We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089-2910, United States of America.

ABSTRACT
We used targeted next generation deep-sequencing (Safe Sequencing System) to measure ultra-rare de novo mutation frequencies in the human male germline by attaching a unique identifier code to each target DNA molecule. Segments from three different human genes (FGFR3, MECP2 and PTPN11) were studied. Regardless of the gene segment, the particular testis donor or the 73 different testis pieces used, the frequencies for any one of the six different mutation types were consistent. Averaging over the C>T/G>A and G>T/C>A mutation types the background mutation frequency was 2.6x10-5 per base pair, while for the four other mutation types the average background frequency was lower at 1.5x10-6 per base pair. These rates far exceed the well documented human genome average frequency per base pair (~10-8) suggesting a non-biological explanation for our data. By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles. Finally, we looked at a previously studied disease mutation in the PTPN11 gene and could easily distinguish true mutations from the SSS background. We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments.

No MeSH data available.


Related in: MedlinePlus

Spurious super-mutants due to very large UID families.One very large UID family on the top row is erroneously counted as twelve additional families. The next nine rows show families with UID sequences that differ at one site from the family in the top row, most likely due to a mistake during PCR amplification. The bottom three rows show families with the same long UID sequence but a different short UID sequence from the family in the top row, most likely due to PCR jumping. All of these families contain the same A>T/T>A super-mutant at the read position 10 bases from the end of the UID (p.10A>T) erroneously increasing its frequency.
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pone.0158340.g005: Spurious super-mutants due to very large UID families.One very large UID family on the top row is erroneously counted as twelve additional families. The next nine rows show families with UID sequences that differ at one site from the family in the top row, most likely due to a mistake during PCR amplification. The bottom three rows show families with the same long UID sequence but a different short UID sequence from the family in the top row, most likely due to PCR jumping. All of these families contain the same A>T/T>A super-mutant at the read position 10 bases from the end of the UID (p.10A>T) erroneously increasing its frequency.

Mentions: There are several ways that spurious super-mutants can be created during the second round of PCR. These ways are most likely to be associated with the creation of a very large UID family. Fig 5 illustrates examples from the PTPN11 data. All of the UID families depicted in the figure have an A>T/T>A super-mutant at position 10. The first UID family listed is very large, containing 15,298 reads. The next nine UID families are much smaller, containing 3 or 4 reads, and each of their UID sequences differ from the UID sequence of the first family at exactly one nucleotide position. These nine UID families are most likely created by mistakes in PCR amplification of the UID sequence of the first family (see calculations #2 and #3 in S1 Text), and should not be counted as distinct families.


Estimating Exceptionally Rare Germline and Somatic Mutation Frequencies via Next Generation Sequencing.

Eboreime J, Choi SK, Yoon SR, Arnheim N, Calabrese P - PLoS ONE (2016)

Spurious super-mutants due to very large UID families.One very large UID family on the top row is erroneously counted as twelve additional families. The next nine rows show families with UID sequences that differ at one site from the family in the top row, most likely due to a mistake during PCR amplification. The bottom three rows show families with the same long UID sequence but a different short UID sequence from the family in the top row, most likely due to PCR jumping. All of these families contain the same A>T/T>A super-mutant at the read position 10 bases from the end of the UID (p.10A>T) erroneously increasing its frequency.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920415&req=5

pone.0158340.g005: Spurious super-mutants due to very large UID families.One very large UID family on the top row is erroneously counted as twelve additional families. The next nine rows show families with UID sequences that differ at one site from the family in the top row, most likely due to a mistake during PCR amplification. The bottom three rows show families with the same long UID sequence but a different short UID sequence from the family in the top row, most likely due to PCR jumping. All of these families contain the same A>T/T>A super-mutant at the read position 10 bases from the end of the UID (p.10A>T) erroneously increasing its frequency.
Mentions: There are several ways that spurious super-mutants can be created during the second round of PCR. These ways are most likely to be associated with the creation of a very large UID family. Fig 5 illustrates examples from the PTPN11 data. All of the UID families depicted in the figure have an A>T/T>A super-mutant at position 10. The first UID family listed is very large, containing 15,298 reads. The next nine UID families are much smaller, containing 3 or 4 reads, and each of their UID sequences differ from the UID sequence of the first family at exactly one nucleotide position. These nine UID families are most likely created by mistakes in PCR amplification of the UID sequence of the first family (see calculations #2 and #3 in S1 Text), and should not be counted as distinct families.

Bottom Line: These rates far exceed the well documented human genome average frequency per base pair (~10-8) suggesting a non-biological explanation for our data.By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles.We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Computational Biology Program, University of Southern California, Los Angeles, CA 90089-2910, United States of America.

ABSTRACT
We used targeted next generation deep-sequencing (Safe Sequencing System) to measure ultra-rare de novo mutation frequencies in the human male germline by attaching a unique identifier code to each target DNA molecule. Segments from three different human genes (FGFR3, MECP2 and PTPN11) were studied. Regardless of the gene segment, the particular testis donor or the 73 different testis pieces used, the frequencies for any one of the six different mutation types were consistent. Averaging over the C>T/G>A and G>T/C>A mutation types the background mutation frequency was 2.6x10-5 per base pair, while for the four other mutation types the average background frequency was lower at 1.5x10-6 per base pair. These rates far exceed the well documented human genome average frequency per base pair (~10-8) suggesting a non-biological explanation for our data. By computational modeling and a new experimental procedure to distinguish between pre-mutagenic lesion base mismatches and a fully mutated base pair in the original DNA molecule, we argue that most of the base-dependent variation in background frequency is due to a mixture of deamination and oxidation during the first two PCR cycles. Finally, we looked at a previously studied disease mutation in the PTPN11 gene and could easily distinguish true mutations from the SSS background. We also discuss the limits and possibilities of this and other methods to measure exceptionally rare mutation frequencies, and we present calculations for other scientists seeking to design their own such experiments.

No MeSH data available.


Related in: MedlinePlus