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  • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
  • C12Q2537/143—Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
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  • C12Q2563/179—Nucleic acid detection characterized by the use of physical, structural and functional properties the label being a nucleic acid

Definitions

• a purification step can be performed, and then an adapter ligation step can be performed on the purified multiplexed PCR amplicons, where the fully ligated molecules are 100% functional and non-directional, similar to Y-shaped adapters, although the method uses separate 5' and 3' adapters that independently ligate to the 5’ and 3’ ends of each strand of the target specific amplicons.
• an adapter ligation step can be performed on the purified multiplexed PCR amplicons, where the fully ligated molecules are 100% functional and non-directional, similar to Y-shaped adapters, although the method uses separate 5' and 3' adapters that independently ligate to the 5’ and 3’ ends of each strand of the target specific amplicons.
• the Swift multiplexed PCR step can be performed as previously disclosed without any modification and is summarized below for reference, utilizing a plurality of target- specific primer pairs each comprising a 5’ tail sequence comprising a universal adapter sequence, and a universal primer complementary to the universal adapter sequence, where the universal primer contains a modification making it susceptible to cleavage by an endonuclease which is required for the subsequent adapter ligation step.
• the PCR utilizes a high fidelity proofreading polymerase that is tolerant of the modified base, where the first PCR cycles have elongated annealing times to allow the high complexity of target- specific primer pairs, each of which is at a low concentration, to create universal adapter tagged amplicons from their target sequences.
• a purification step can be performed and then library quantification, pooling and sequencing can be performed.
• This novel method both increases library yield and results in libraries enriched for fully ligated, functional molecules with double- stranded adapters that can easily be quantified by fluorometric methods such as Qubit, or electrophoretic chips such as Bioanalyzer, in addition to qPCR.
• fluorometric methods such as Qubit, or electrophoretic chips such as Bioanalyzer
• an additional purification step is avoided that would typically be required between adapter ligation and PCR, as well as avoiding addition of PCR reagents following the ligation reaction if a purification step was not required.
• the workflow preserves the original workflow of two enzymatic reactions and two purification steps.
• the starting quantity must be greater than the target quantity, so the number of PCR cycles applied must achieve a library yield that is greater than the target quantity.
• a bead-based purification step is performed.
• PCR thermocycling is then performed for the required number of cycles using the forward and reverse primer to achieve the desired yield of double stranded product.
• the splint oligonucleotides comprise a 3’ blocking group to prevent priming activity during PCR, and oligonucleotide subunits require a 5’ phosphate for splint ligation.
• the subunits can be added to the reaction at a concentration too low to support PCR so that only the forward and reverse primers amplify the product to prevent unused subunit oligonucleotides from truncating the fully assembled product by priming during PCR.
• the primer corresponding to the 5’ adapter can be used as both an adapter for ligation and a primer for PCR amplification in the same ligation-coupled- PCR reaction.
• an optional blocker oligonucleotide that is complementary to the 3’ end of the indexing primer that corresponds to the 3’ adapter may be pre-annealed to this primer. The blocker oligonucleotide prevents this primer from participating in the initial 5’ adapter ligation step at the first incubation temperature but has a T m below the PCR annealing temperature or is inactivated so it does not block priming activity during PCR (see Figures 2B- 2G and 4A-4E).
• FIGURE II depicts a comparison of adapter-dimer formation in different NGS library protocols.
• FIGURE 2 A depicts an exemplary workflow where a partially double- stranded DNA substrate is produced by endonuclease cleavage which yields 3’ overhangs, followed by 5’ adapter ligation and indexing PCR using two indexing primers which each have a 3’ terminal portion that is complementary to a 5’ portion of the 3’ overhangs (a first common nucleotide sequence), where either of the indexing primers can function as the 5’ adapter.
• FIGURE 3C further depicts that the stem loop truncated adapter 5 also comprises a non-replicable modification represented by the black circle within the loop sequence so formation of completely replicated hairpin products are prevented.
• FIGURE 6C depicts assembly of a TruSeq Illumina indexing primer with the (T)i2(rU)4 (SEQ ID NO: 1) sequence compatible with downstream enzymatic normalization.
• FIGURE 6C discloses SEQ ID NOs: 113- 118, and 116-117, respectively, in order of appearance.
• FIGURE 6D discloses SEQ ID NOs: 113-117, 119-122, 83, 82, 93, 118, 116-118, 116-117, 93, 123, 122, 83, and 82, respectively, in order of appearance.
• FIGURE 10E depicts the Picard plots for NGS libraries described in Example 6 (NGS library prep B 250 ng).
• FIGURE 11 A depicts a Bio Analyzer trace for libraries prepared from pictogram amount of DNA by methods described in Example 6.
• the PCR substrate is a truncated NGS library comprising a first adapter with cleavable bases, where an endonuclease cleaves one strand of the first adapter to enable annealing and ligation of the second adapter.
• the PCR primer is also assembled by ligation of tandem oligonucleotides linked by complementary splint oligonucleotides.
• FIGURE 1A depicts all steps from endonuclease cleavage through indexing PCR occurring in a single closed tube, however, it should be understood that the steps from 5’ adapter ligation through indexing PCR may be performed in a single closed tube regardless of if endonuclease cleavage (or other enzymatic processing) is also performed.
• the cleavage steps to yield the partially double-stranded DNA substrate can occur in a single closed tube with the ligation and PCR or can be separately performed before the ligation-coupled PCR.
• the enzymatic processing to obtain the partially double-stranded DNA substrate can be performed in a prior step or can be performed as a part of the workflow in a single closed tube as shown in FIGURE 1A.
• the final reaction combines endonuclease cleavage of oligonucleotide 8 by USER enzyme, ligation of the truncated 5’ hairpin adapter 6 to the 5’ end of DNA after annealing to the reverse complement of the 3’ adapter oligonucleotide 7, which is followed by indexing PCR to amplify the library and complete the adapter sequences, in a single closed tube as shown on Figure 1H.
• Secondary structure of the hairpin 5’ adapter prevents its activity as a primer during PCR so that it does not truncate the 5’ adapter from completed library molecules.
• UDG enzyme does not produce breaks within the oligonucleotide 1 but creates a number of abasic sites that are sufficient for the reduction of the annealing temperature below 37°C and dissociation of the oligonucleotide 8 from the complementary 3’ adapter oligonucleotide 7 to allow annealing and ligation of the 5’ adapter indexing primer 3 to the DNA end (FIGURE 1G).
• a linear blocker 5 that is pre-annealed to the full- length 3’ adapter indexing primer 4 prior to being added to the ligation reaction is used (Fig. 2B). It prevents the 3’ terminus of this primer from being available to ligate to the amplicon substrates after annealing to sequence 2 at the ligation incubation temperature.
• the blocker T m is lower than the PCR annealing temperature or the blocker is inactivated, so the 3’ adapter indexing primer 4 can efficiently anneal during PCR.
• Fig. 2C a linear blocker 5 that is pre-annealed to the full- length 3’ adapter indexing primer 4 prior to being added to the ligation reaction is used
• a linear blocker comprising a low T m so its inactive during PCR or (b) a hairpin blocker that becomes inactivated during PCR when using Illumina TruSeq adapters.
• An G7 indexing primer 4 and a linear blocker 5a with a lower T m or a hairpin blocker 5b that is inactivated so annealing to the 3’ adapter indexing primer is permitted during the ligation reaction but does not anneal during PCR:
• the linear blocker 5a comprises one or more mismatches to the universal adapter sequence on indexing primer 4 (3 mismatches are shown) or comprises an insertion of non complementary bases including but not limited to one or more T deoxynucleotides (6 are shown), either of which reduce its T m below the PCR annealing temperature so that the blocker cannot block priming of the indexing primer.
• the hairpin adapter 5 depicts a solution to this problem where a hairpin truncated adapter 5 is used for ligation-coupled-PCR when using indexing primers 3 and 4 that lack the common adapter sequence at the 3’ ends.
• the endonuclease cleavage is followed by annealing and ligation of the 3’ single- stranded overhang of the hairpin adapter which comprises at least a portion of the common adapter sequence.
• the hairpin adapter also comprises a unique sequence of the 5’ adapter and the reverse complement of this sequence at the 5’ end of the blocker to create a hairpin also comprising a T loop sequence.
• the truncated 5’ adapter relative to a full length adapter can either be linear (b) or comprise a hairpin with a stem loop structure (c), where the hairpin prevents adapter annealing and extension during PCR due to competition with its stable self-complementarity. This prevents the hairpin adapter from truncating completed 5’ adapters during PCR, making it the preferred embodiment for a truncated 5’ adapter.
• the linear truncated adapter can efficiently anneal, extend, and truncate completed 5’ adapter molecules during PCR which reduces the amplified library yield.
• the ligation-coupled-PCR can include DNA subunits for ligation, a ligase, a polymerase, a primer pair and a substrate for PCR amplification that may be products of the DNA subunit ligation, and optionally an endonuclease.
• the polymerase can be a thermostable hot-start DNA polymerase such as Taq DNA Polymerase or preferably, for NGS library amplification, the polymerase is a high fidelity polymerase with 3 ’-5’ exonuclease proofreading activity.
• a method for ligation-coupled PCR includes: (i) providing a partially double-stranded DNA substrate comprising a first strand and a second strand, the partially double-stranded DNA substrate including a first 3’ overhang, a double-stranded portion, and a second 3’ overhang, where the first strand includes, in a 5’ to 3’ direction, a first 5’ end, a first portion and a second portion, where the second strand includes, in a 5’ to 3’ direction, a second 5’ end, a third portion and a fourth portion, where the first portion of the first strand and the third portion of the second strand are complementary and form the double-stranded portion, where the second portion of the first strand forms the first 3’ overhang, where the fourth portion of the second strand forms the second 3’ overhang, and where the second portion of the first strand and the fourth portion of the second strand each include a first common nucleotide sequence positioned at
• the second portion of the first strand and the fourth portion of the second strand can each include from about 4 bases to about 100 bases.
• the first common nucleotide sequence and the first 3’ terminal portion can have a melting temperature (T m ) greater than the ligation temperature.
• T m melting temperature
• the melting temperature of the first common nucleotide sequence and the first 3’ terminal portion can be 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, or 40 °C higher than the ligation temperature.
• the blocker oligonucleotide can further include a hairpin portion positioned 3’ to the first additional portion where the hairpin portion includes a first hairpin sequence and a second hairpin sequence, the first hairpin sequence being positioned 5’ to the second hairpin sequence, where the first hairpin sequence and the second hairpin sequence are complementary, and where the hairpin portion has a melting temperature greater than the first annealing temperature, the second annealing temperature, and the third annealing temperature.
• the blocker oligonucleotide further include a 3’ hydroxyl group.
• the hairpin portion can further include a third hairpin sequence between the first hairpin sequence and the second hairpin sequence.
• the third sequence can form a loop sufficient to allow formation of a stable stem-loop structure by the hairpin portion and the first additional portion.
• the third hairpin sequence can have a length from about 4 to about 20 bases or more. It should be understood that the third hairpin sequence can form a loop sufficient to allow formation of a stable stem- loop structure with the first and second hairpin sequences.
• a melting temperature of the hairpin portion can be greater than a melting temperature between the 5’ portion and each of the plurality of second indexing primers, the additional portion and each of the plurality of second indexing primers or both.
• each of the plurality of second indexing primers can have a length of from about 20 bases to about 100 bases.
• each of the plurality of second indexing primers can have a length from about 20 bases to about 100 bases, about 20 bases to about 90 bases, about 20 bases to about 80 bases, about 20 bases to about 70 bases, about 20 bases to about 60 bases, about 20 bases to about 50 bases, about 20 bases to about 40 bases, about 20 bases to about 30 bases, about 25 bases to about 100 bases, about 25 bases to about 90 bases, about 25 bases to about 80 bases, about 25 bases to about 70 bases, about 25 bases to about 60 bases, about 25 bases to about 50 bases, about 25 bases to about 40 bases, about 25 bases to about 30 bases, about 30 bases to about 100 bases, about 30 bases to about 90 bases, about 30 bases to about 80 bases, about 30 bases to about 70 bases, about 30 bases to about 60 bases,
• the first extension duration, the second extension duration, the third extension duration, and the fourth extension duration can each be from about 30 seconds to about 5 minutes. It should be understood that the first extension temperature, the second extension temperature, and the third extension temperature and the first extension duration, the second extension duration, and the third extension duration should be sufficient for the extension required in each respective PCR step to occur.
• One of skill in the art can readily determine such temperature and the necessary time through routine experimentation and/or knowledge in the art.
• the method can further include sequencing the seventh strand and the ninth strand or the eighth strand and the tenth strand.
• the method can further include sequencing the seventh strand and the ninth strand or the eighth strand and the tenth strand.
• the enzyme capable of cleaving the inosine bases is Endonuclease V.
• the partially double-stranded DNA substrate can be produced by: providing a fragmented DNA substrate molecule, performing end repair to yield a blunt-ended, double- stranded starting DNA substrate molecule which has a first starting strand a a second starting strand each with a 5’ end a 3’ end, adding a plurality of 3’ adapter strands where each 3’ adapter strand is annealed to a complementary strand which includes dU bases and a ligase to the blunt-ended, double stranded starting DNA molecule, incubating the plurality of 3’ adapter strands annealed to complementary strands including dU bases, the ligase, and the blunt- ended double- stranded DNA substrate molecule under conditions sufficient to ligate one of the plurality of 3’ adapter strands to each 3’ end of the
• a method for ligation-coupled PCR can include (i) providing a double- stranded DNA substrate comprising a first strand and a second strand, the first strand comprising a first 3’ terminal portion and a first 5’ terminal portion and the second strand comprising a second 3’ terminal portion and a second 5’ terminal portion; (ii) adding a ligase, a first oligonucleotide, a second oligonucleotide, a third oligonucleotide, a fourth oligonucleotide, a fifth oligonucleotide, a sixth oligonucleotide, a DNA polymerase and deoxynucleotide triphosphates (dNTPs) to the double-stranded DNA substrate to yield a first reaction mixture, the first oligonucleotide comprises a third 3’ terminal portion complementary to at least a portion of the first 3’ terminal portion of the
• the annealing temperature can be from about 55 °C to about 65 °C.
• the annealing temperature can each be from about 55 °C to about 65 °C, 55 °C to about 60 °C , about 60 °C to about 65 °C, about 55 °C, 56 °C, 57 °C, 58 °C, 59 °C, 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, or 65 °C.
• the first strand can be ligated on one of the partially double-stranded DNA substrates while the second strand can be ligated on another, in such an instance the third oligonucleotide and fourth oligonucleotide could still be formed.
• the library was quantified using the Kapa qPCR Assay Kit for Illumina before normalization was completed via the Swift Normalase kit in accordance with the manufacturer’s workflow recommendations to achieve a 2nM pool.
• the pool was loaded onto an Illumina MiniSeq and sequenced with paired end reads of 151 bases.
• Lanes (H), (I), and (J) demonstrate over 90% ligation of modified indexing oligo using T3 DNA ligase incubated in reaction conditions optimized for PCR without and with HiFi DNA polymerase added after and before incubation for 15 min at 25 °C, respectively.
• the 3' adapter utilized in this example is a blunt- ended truncated adapter (domain 2); whereas, the 5' adapter is either a truncated hairpin (domain 5 in Fig. 3B) with a relatively short single stranded 3' portion or a truncated linear adapter (domain 5 in Fig. 3A) with substantially higher T m .
• Melting temperature of the stem region of the hairpin 5’ adapter used in this example is very high (T m ⁇ 80°C) so that during primer annealing and extension the hairpin 5’ adapter is folded and does not participate in PCR due to low T m of its single stranded 3’ portion (T m ⁇ 50°C).
• T4 DNA Ligase (Rapid) (Enzymatics cat# L6030-HC-L)

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