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

Eboreime, Jordan, Choi, Soo-Kung, Yoon, Song-Ro, Arnheim, Norman, Calabrese, Peter·2016·PLOS ONE
DOI 10.1371/journal.pone.0158340PMC PMC4920415
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Figures

Fig 1 First PCR Round of the SSS strategy. a. Two primers (forward and reverse) are used for the first two SSS PCR cycles (all primers are listed in S1 Fig). Each primer (5’ to 3’) contains a Sequencing primer region (SEQ), a Unique Identifier (UID) and the forward (GENEF) or reverse (GENER) versions of the gene specific DNA sequences. Only the reverse primer contains a barcode for multiplex analysis (BC). b. Duplex genomic DNA fragment carrying a gene target that experienced an in vivo germline mutation (red circle). c. The two new daughter strands of the first denaturation, primer hybridization and primer extension. The F42 and R28 represent one of the many UIDs on the forward and reverse primers, respectively. The blue arrow indicates the direction of the reverse primer extension that includes the specific target region (blue dashes) and extends through the target as does the purple forward primer on the other strand (purple dashes). The green overhang at the extreme 5’ ends will anneal to the primers used in the second PCR round. Two different extension products are created (#1 and #2). Notice that extension product #2, with the already existing in vivo mutation is (for convenience only) also shown to experience a new in vitro mutation (black circle). In the subsequent steps, for simplicity, we only follow one of the two extension products (#2). d. The second cycle with the same primer mix, invariably involves a primer (black) with a different UID (R12) and copies DNA strand #2 (black dashes). Subsequent single strand-specific exonuclease digestion removes all the initial PCR primers, trims the 3’ DNA overhangs from the second cycle primer extension products and any remaining single-stranded genomic DNA. e. Importantly, F42-R12 is tagged by different UIDs at each end and is then subjected to the second round of PCR (see Fig 2).
Fig 1 First PCR Round of the SSS strategy. a. Two primers (forward and reverse) are used for the first two SSS PCR cycles (all primers are listed in S1 Fig). Each primer (5’ to 3’) contains a Sequencing primer region (SEQ), a Unique Identifier (UID) and the forward (GENEF) or reverse (GENER) versions of the gene specific DNA sequences. Only the reverse primer contains a barcode for multiplex analysis (BC). b. Duplex genomic DNA fragment carrying a gene target that experienced an in vivo germline mutation (red circle). c. The two new daughter strands of the first denaturation, primer hybridization and primer extension. The F42 and R28 represent one of the many UIDs on the forward and reverse primers, respectively. The blue arrow indicates the direction of the reverse primer extension that includes the specific target region (blue dashes) and extends through the target as does the purple forward primer on the other strand (purple dashes). The green overhang at the extreme 5’ ends will anneal to the primers used in the second PCR round. Two different extension products are created (#1 and #2). Notice that extension product #2, with the already existing in vivo mutation is (for convenience only) also shown to experience a new in vitro mutation (black circle). In the subsequent steps, for simplicity, we only follow one of the two extension products (#2). d. The second cycle with the same primer mix, invariably involves a primer (black) with a different UID (R12) and copies DNA strand #2 (black dashes). Subsequent single strand-specific exonuclease digestion removes all the initial PCR primers, trims the 3’ DNA overhangs from the second cycle primer extension products and any remaining single-stranded genomic DNA. e. Importantly, F42-R12 is tagged by different UIDs at each end and is then subjected to the second round of PCR (see Fig 2).CC BY · PubMed Central
Fig 2 Second Round of the SSS Strategy. a. Universal forward and reverse primers are used to amplify the product made in the first round of PCR (see Fig 1E). The primers contain the Illumina Sequencing primer (ISP), which is partially complementary to the Sequencing primer region of the primers used in the first two PCR cycles (Fig 1A). The universal primers also contain the complement of the Flow cell grafting sequence (FC). b. Products of the first few cycles of the second round using the template shown in Fig 1E. c. Further amplification for 28–30 additional cycles leads to the creation of UID families of which the one shown is representative. The original genomic mutation and the mutation that arose during the first two SSS PCR cycles are both present in this particular family. Notice that one of the family members has accumulated an additional in vitro mutation (black star) due to events during the second PCR round or the final sequencing step. d. Analysis of the UID family is able to eliminate the latter error from consideration.
Fig 2 Second Round of the SSS Strategy. a. Universal forward and reverse primers are used to amplify the product made in the first round of PCR (see Fig 1E). The primers contain the Illumina Sequencing primer (ISP), which is partially complementary to the Sequencing primer region of the primers used in the first two PCR cycles (Fig 1A). The universal primers also contain the complement of the Flow cell grafting sequence (FC). b. Products of the first few cycles of the second round using the template shown in Fig 1E. c. Further amplification for 28–30 additional cycles leads to the creation of UID families of which the one shown is representative. The original genomic mutation and the mutation that arose during the first two SSS PCR cycles are both present in this particular family. Notice that one of the family members has accumulated an additional in vitro mutation (black star) due to events during the second PCR round or the final sequencing step. d. Analysis of the UID family is able to eliminate the latter error from consideration.CC BY · PubMed Central
Fig 3 PTPN11 mutation frequencies color-coded by reference base. Each dot represents the average of 9 different libraries from three testis pieces. Red indicates an A or T base, blue indicates a C or G (non-CpG) base, and green indicates a CpG. The mutation frequency is the sum of all mutations at that site, so, e.g., if a site is a C, the mutation frequency is the sum of the C>A, C>G, and C>T frequencies at that site. The 95% confidence interval for each position is also shown. The data on one base pair (at position 80; c.922) has not been included since it has a much greater mutation frequency; for an explanation, see below.
Fig 3 PTPN11 mutation frequencies color-coded by reference base. Each dot represents the average of 9 different libraries from three testis pieces. Red indicates an A or T base, blue indicates a C or G (non-CpG) base, and green indicates a CpG. The mutation frequency is the sum of all mutations at that site, so, e.g., if a site is a C, the mutation frequency is the sum of the C>A, C>G, and C>T frequencies at that site. The 95% confidence interval for each position is also shown. The data on one base pair (at position 80; c.922) has not been included since it has a much greater mutation frequency; for an explanation, see below.CC BY · PubMed Central
Fig 4 Separate method and pre-existing mismatches. The Separate method separately amplifies the coding and non-coding strands. The aliquot on the left-hand side only amplifies the top strand, while the aliquot on the right-hand side only amplifies the bottom strand. The yellow rectangles indicate strands that are not primer extension products and are removed by exonucleases. For the original molecule shown with a pre-existing U:G mismatch, the Separate method detects a C>T mutation on the left-hand side and no mutation on the right-hand side.
Fig 4 Separate method and pre-existing mismatches. The Separate method separately amplifies the coding and non-coding strands. The aliquot on the left-hand side only amplifies the top strand, while the aliquot on the right-hand side only amplifies the bottom strand. The yellow rectangles indicate strands that are not primer extension products and are removed by exonucleases. For the original molecule shown with a pre-existing U:G mismatch, the Separate method detects a C>T mutation on the left-hand side and no mutation on the right-hand side.CC BY · PubMed Central
Fig 5 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.
Fig 5 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.CC BY · PubMed Central

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Citations

Cites

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Metadata

Authors
Eboreime, J., Choi, S.-K., Yoon, S.-R., Arnheim, N., & Calabrese, P.
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2016-06-24
Volume
11
Issue
6
Pages
e0158340
ISSN
1932-6203
DOI
10.1371/journal.pone.0158340
PMC
PMC4920415
Cited by
11
References
1
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
National Institute of General Medical Sciences (RO1 GM-36745)

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