`
` Published online September 30, 2004
`
`Nucleic Acids Research, 2004, Vol. 32, No. 17
`e135
`doi:10.1093/nar/gnh132
`
`Molecular barcodes detect redundancy and
`contamination in hairpin-bisulfite PCR
`Brooks E. Miner1,*, Reinhard J. Sto¨ ger1, Alice F. Burden1, Charles D. Laird1,2
`and R. Scott Hansen3
`
`1Department of Biology, 2Department of Genome Sciences and 3Department of Medicine,
`University of Washington, Seattle, WA 98195, USA
`
`Received August 12, 2004; Revised and Accepted September 11, 2004
`
`ABSTRACT
`
`PCR amplification of limited amounts of DNA template
`carries an increased risk of product redundancy and
`contamination. We use molecular barcoding to label
`each genomic DNA template with an individual
`sequence tag prior to PCR amplification. In addition,
`we include molecular ‘batch-stamps’ that effectively
`label each genomic template with a sample ID and
`analysis date. This highly sensitive method identifies
`redundant and contaminant sequences and serves as
`a reliable method for positive identification of desired
`sequences; we can therefore capture accurately the
`genomic template diversity in the sample analyzed.
`Although our application described here involves
`the use of hairpin-bisulfite PCR for amplification
`of double-stranded DNA, the method can readily be
`adapted to single-strand PCR. Useful applications will
`include analyses of limited template DNA for bio-
`medical, ancient DNA and forensic purposes.
`
`INTRODUCTION
`
`The polymerase chain reaction (PCR) allows multiple copies
`of selected DNA sequences to be copied from limited amounts
`of DNA template (1). Reactions with limited template, how-
`ever, increase the risk of amplifying contaminant DNA and
`can also result in a skewed yield of PCR products such that
`there is a high degree of redundancy for a small portion of the
`original genomic sequences (2). Redundancy can either be
`useful, e.g. in tracking mutations arising during the PCR
`amplification of individual molecules, or unwelcome, for
`example, when the goal is to compare and quantify sequences
`from different cells represented in the same DNA sample, as in
`bisulfite methylation analysis (3). The frequent observation of
`multiple amplified sequences derived from a single original
`molecule was also noted by Millar et al. (4) in the context of
`bisulfite genomic sequencing, a method increasingly used in
`epigenetic research.
`In response to the challenges of PCR redundancy and con-
`tamination associated with PCR amplification of limited
`
`amounts of DNA template, we have labeled genomic DNA
`fragments with molecular sequence barcodes and ‘batch-
`stamps’ prior to PCR amplification. This was accomplished
`by including these molecular labels in the hairpin linker
`sequence that we use in hairpin-bisulfite PCR (5). This
`encoded information enables us to track the genomic origin
`of each sequence obtained from PCR and subsequent bacterial
`cloning. Each genomic fragment is marked prior to amplifica-
`tion, allowing us to identify contaminant and redundant
`sequences and to quantify accurately the proportion of cells
`carrying a particular sequence variant by counting only dis-
`tinctly tagged sequences. This highly sensitive method offers
`confirmation of
`the independent genomic origin of all
`sequences in final data sets derived from PCR amplification.
`
`MATERIALS AND METHODS
`
`Conditions for hairpin-bisulfite PCR of human genomic FMR1
`sequences (5) were as follows: 5 mg of genomic DNA was
`cleaved by 10 U each of restriction endonucleases DraIII and
`AluI for 1 h at 37C, followed by enzyme inactivation at 65C
`for 20 min. The use of a second restriction endonuclease,
`in this case AluI, removed the CG-rich sequence distal to
`the region analyzed. Ligation of the hairpin linker (50P-AGC-
`GATGCDDDDDDDGCATCGCT-TGA, with variations in
`the non-random nucleotides for batch-stamps) to DraIII-
`cleaved genomic DNA was for 15 min at 20C, using 400 U
`of T4 ligase in 20 ml with 1· ligation buffer (New England
`Biolabs), followed by enzyme inactivation at 65C for 20 min.
`The bisulfite conversion followed the protocol of Laird et al.
`(5) with additional thermal denaturation steps. Hairpin-ligated
`DNA was denatured in 0.3 M NaOH for 20 min, then heated to
`100C for 1 min before addition of sodium bisulfite and hydro-
`quinone to 3.4 M and 1 mM, respectively. The reaction mix-
`ture was incubated for 6 h at 55C, with additional thermal
`denaturation steps (99C for 90 s, 10 times over the 6 h), and
`then incubated for an additional 6 h at 55C. This was followed
`by a purification step using QIAquick PCR purification col-
`umns (Qiagen), subsequent treament with NaOH (final con-
`centration 0.3 M) at 37C for 20 min, and another purification
`using Microspin S-200 HR columns (Amersham Pharmacia
`Biosciences). PCR conditions were Hotstar Master Mix
`
`*To whom correspondence should be addressed at Box 351800, Seattle, WA 98195, USA. Tel: +1 206 616 9385; Fax: +1 206 543 3041; Email:
`miner@u.washington.edu
`Correspondence may also be addressed to Charles D. Laird. Email: cdlaird@u.washington.edu
`
`Nucleic Acids Research, Vol. 32 No. 17 ª Oxford University Press 2004; all rights reserved
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`Oxford, Exh. 1007, p. 1
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`(Qiagen), with denaturation at 95C for 15 min, followed by 38
`cycles of denaturing at 95C for 30 s, annealing at 58C for
`30 s, and extension at 72C for 45 s; this was followed by
`a final extension at 72C for 5 min. Primers used were
`(i) first primer, 50-CCTCTCTCTTCAAATAACCTAAAA-
`AC-30 and (ii) second primer, 50-GTTGYGGGTGTAAA-
`TATTGAAATTA-30.
`All PCR products were analyzed by agarose gel electro-
`phoresis; further cloning and sequencing of appropriately
`sized products was with TOPO TA Cloning Kits (Invitrogen
`Life Technologies); sequencing reactions were carried out
`with fluorescent dideoxy nucleotides (BIGDYE Terminator
`3.1, Applied Biosystems), at either the DNA Sequencing
`Facility, Department of Biochemistry, or the Comparative
`Genomics Center, Department of Biology, University of
`Washington. Each sequence was proofread against
`the
`sequence trace; errant base calling was corrected manually
`before being presented here. For purposes of analysis and
`presentation, the output sequence was folded, using word-
`processing software,
`into a hairpin conformation so that
`both strands aligned.
`
`RESULTS
`
`The challenge of amplifying limited amounts of DNA tem-
`plate can result from trace amounts of initial DNA sample, or
`from laboratory analyses that include substantial DNA degra-
`dation as a necessary side effect of processing, as in bisulfite
`genomic sequencing (6). One of
`the major problems
`encountered in these analyses is to capture accurately the
`genomic template diversity following the steps of PCR
`and bacterial cloning. Hairpin-bisulfite PCR involves the
`ligation of a synthetic hairpin linker to the ends of a
`double-stranded genomic DNA fragment prior to bisulfite
`conversion and PCR amplification (5). While the primary
`purpose of the hairpin linker is to maintain attachment of
`complementary strands, it can also be used to encode each
`ligated genomic fragment with information that distinguishes
`it from other sequences within a sample, allowing us to
`evaluate cloned sequences for redundancy and contamina-
`tion. To accomplish this, we replaced the 6 nt loop of our
`hairpin linker (5) with 7 nt randomly selected from A, G
`and T. Cytosine was not used because its identity would be
`ambiguous after bisulfite conversion. With a random 7 nt
`barcode, the number of possible codes is 2187; in selecting
`15 cloned PCR products from one DNA sample, the probab-
`ility that two of these will be different genomic fragments
`labeled with identical 7 nt barcodes is 0.047 (see Supple-
`mentary Material). Some applications will require a larger
`pool of random-sequence barcodes if more independently
`derived sequences are required. We have used linkers with
`up to 13 nt in the hairpin loop with no observable detriments
`to sequence recovery. A 13 nt barcode gives 1.6 · 106
`different codes; even for a selection of 100 cloned PCR
`products, the probability that two of these would be different
`genomic fragments labeled with identical barcodes is only
`0.0031 (see Supplementary Material).
`In addition to adding the random barcode, we ‘batch-
`stamped’ molecules by encoding the hairpin linker with infor-
`mation that would designate the sample analyzed and the
`
`Figure 1. Schematic of barcoded and batch-stamped hairpin linker, designed
`for ligation to DraIII-cut genomic DNA of FMR1. The letter D represents a
`nucleotide randomly selected from A, G and T.
`
`date of analysis. We designed multiple variants of the hairpin
`linker by changing nucleotides in the stem of the linker. These
`stem changes represented different batches of linkers, each of
`which we used for the analysis of a different sample. Thus, the
`resulting sequences each bear a consistent ‘batch-stamp’
`encoded in the stem, and a randomly variable barcode encoded
`in the loop (Figure 1).
`We applied our enhanced hairpin-bisulfite PCR method to
`the FMR1 promoter region in the DNA of males with fragile
`X syndrome. The classes of sequences recovered included
`hypermethylated sequences with distinctive barcodes and pat-
`terns of methylation (Figure 2a), redundant hypermethylated
`sequences with identical barcodes and methylation patterns
`(Figure 2b–c), hypomethylated sequences with distinctive
`barcodes (Figure 2d), redundant hypomethylated sequences
`with identical barcodes (Figure 2e–f), and contaminant
`sequences with our original linker that predates the barcoding
`(Figure 2g). The number of sequences cloned influenced the
`observed proportion of redundancy among the recovered
`sequences;
`the observed proportions of both redundancy
`and contamination appeared to depend on the initial amount
`of DNA used and the quality of the bisulfite conversion.
`Among eight different DNA samples analyzed, the propor-
`tion of sequences that were redundant ranged from 7 to 51%,
`and the proportion of sequences that were contaminants ran-
`ged from 0 to 14%. Occasionally, contaminant sequences
`were cloned from PCR reactions in which control reactions
`(those without template DNA) showed no DNA bands on
`ethidium–bromide-stained agarose gels. In these contexts,
`barcoding serves as a highly accurate method for positive
`identification of desired sequences.
`Within 142 barcodes recovered from multiple reactions with
`FMR1, the average nucleotide composition was 54% T, 26% G
`and 19% A. This bias is similar to that previously reported for
`the influence of loop nucleotides on the stability of DNA
`hairpin structures (7).
`
`DISCUSSION
`
`The concept of molecular barcoding has previously been
`used in signature-tagged mutagenesis (8,9), to track the ori-
`gins of expressed sequence tags (10), and to label objects for
`identification and authentication (11,12). Here, we apply this
`concept to the labeling of individual genomic fragments with
`distinct sequence tags. The ability to barcode and ‘batch-
`stamp’ genomic DNA sequences from individual alleles is
`useful in situations where template DNA is limited, thus
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`Nucleic Acids Research, 2004, Vol. 32, No. 17
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`e135
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`Figure 2. FMR1 promoter sequences, with inferred methylation states of CpG sites, recovered from male fragile X patients using hairpin-bisulfite PCR with linker
`barcoding and batch-stamping. Methods are as described in the text. Unconverted (methylated) CpG dyads are black, and converted (unmethylated) CpG dyads are
`boxed. Within the 26 nt linker (boxed region at left), the randomized 7 nt variable barcodes are shaded at far left; the designated variable batch-stamps (A:T or T:A) are
`shaded at right. All sequences show 100% conversion of non-CpG cytosines. (a) A distinctive hypermethylated sequence. (b and c) Redundant hypermethylated
`sequences recovered from independent bacterial colonies, with identical barcodes and methylation patterns. (d) A hypomethylated sequence with a distinctive
`barcode. (e and f) Redundant hypomethylated sequences with identical barcodes recovered from independent bacterial colonies. These are distinguishable as
`redundant and as different from the hypomethylated sequence ‘d’ only because of barcoding. (g) A contaminant sequence bearing our original hairpin linker that
`predates the addition of the barcode and batch-stamp. This sequence was recovered during analysis of the same sample that generated sequences ‘a–c’. Sequences
`‘a–c’ carry a different batch-stamp than sequences ‘d–f’, with the inversion of the A–T base pair, confirming that these sequence sets came from different DNA
`samples. Redundant hypermethylated sequences are denoted with asterisks (*), and redundant hypomethylated sequences with plus signs (+).
`
`identifying contaminants and redundant sequences arising
`from template re-cloning. We have identified contaminant
`sequences even when multiple control (no DNA) PCR sam-
`ples were negative. Barcoding allows for quantification of the
`relative abundance of genomic methylation patterns or poly-
`morphic sequences by correcting for skewing that can arise
`from PCR amplification or the cloning of the products. The
`barcoding method thus provides a definitive solution to the
`problem identified by both Taylor et al. (2) and Millar et al.
`(4), in which multiple amplified sequences are derived from
`a single original molecule when template DNA is limited or
`of poor quality. The method also allows for the analysis of
`mutations arising during PCR amplification. Although our
`application described here involves the use of hairpin-bisul-
`fite PCR for amplification of double-stranded DNA,
`the
`method can readily be adapted to single-strand PCR. Useful
`applications will include analyses of limited template DNA
`for biomedical, ancient DNA and forensic purposes.
`
`SUPPLEMENTARY MATERIAL
`
`Supplementary Material is available at NAR Online.
`
`ACKNOWLEDGEMENTS
`
`We thank Stanley Gartler, Diane Genereux and Carl Bergstrom
`for helpful discussions and suggestions. Support was provided
`by National Institutes of Health Grants GM 53805, HD 02274
`and HD 16659.
`
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