{"id":1606,"date":"2017-11-30T14:47:42","date_gmt":"2017-11-30T14:47:42","guid":{"rendered":"https:\/\/www.mybiosource.com\/learn\/?page_id=1606"},"modified":"2023-03-02T12:02:08","modified_gmt":"2023-03-02T12:02:08","slug":"genome-library-paired-end-sequencing","status":"publish","type":"page","link":"https:\/\/www.mybiosource.com\/learn\/testing-procedures\/genome-library-paired-end-sequencing\/","title":{"rendered":"Genome Library – Paired End Sequencing"},"content":{"rendered":"

Introduction <\/strong><\/p>\n

This is a protocol for preparing genomic library for paired end sequencing. Sequencing library are produced by adding adapter sequences onto the ends of the DNA<\/span> fragments. These chemically synthesized single stranded or double stranded oligonucleotide can be joined to other DNA<\/span> or RNA molecules.<\/p>\n

Sample Prep Workflow <\/strong><\/h3>\n

The input genomic DNA<\/span> is first fragmented by hydrodynamic shearing, a process commonly applied to get a precise DNA<\/span> fragment size. The final input fragments should be less than 800 bp in size. These fragments are phosphorylated and blunt ended. A single A’ nucleotide is added to the 3′ end of the fragments. This is done to aid the ligation of the adapter. Adapter ligation occurring at both ends of the genomic DNA sequence<\/span> confers different sequences. The product of the ligation are purified and separated based on size by gel electrophoresis<\/span>. The resulting strands are amplified using PCR<\/span> (Polymerase<\/span> Chain Reaction). The resulting sample library is again purified and size selected by gel electrophoresis<\/span>. The final product is quantified prior to seeding clusters on a flow cell.<\/p>\n

\u00a0Liquid Handling <\/strong><\/h3>\n

Small differences in volumes can give rise to significant difference in cluster numbers. So good liquid handling measures are essential. Pipetting small volumes can also lead<\/span> to the error in standard curves and qPCR. When handling solutions of high molecular weight, care should be taken for evenly dispersed solution. Pipetting errors can be minimized if large number of samples are prepared at a time.<\/p>\n

Potential DNA Contaminants<\/strong><\/h3>\n

Interference of the DNA<\/span> contaminants may result in incorrect quantification<\/span> of DNA<\/span>. These DNA<\/span> contaminants may be a RNA, small nucleic acid fragments, nucleotides<\/span>, single\u2010stranded DNA<\/span>, excess proteins<\/span>, or other contaminating materials. DNA<\/span> quality also affects the quantity of usable DNA<\/span>. If the DNA<\/span> present in the sample is heavily damaged, the number of fragments available for DNA<\/span> library production may change. High molecular weight dsDNA also interfere in DNA<\/span>\u00a0 quantification<\/span>. Bacterial<\/span> artificial chromosomes (BACs) even with best efforts of purification contains some parts of the host DNA<\/span>. These DNA<\/span> molecules eventually will produce unwanted clusters. This DNA<\/span> contamination can be countered by quantifying aligned reads against a known bacterial<\/span> sequences and subtracting these out. High molecular weight contamination is estimated prior to library preparation by qPCR designed for chromosmal markers<\/span>.<\/p>\n

Temperature Considerations <\/strong><\/h3>\n

In steps of adapter ligation, temperature elevation should be avoided. DNA<\/span> molecules which AT denatures quickly than DNA<\/span> molecules containing GC, this sets a bias for the experiment.\u00a0 As a general rule, libraries should be kept at temperatures below <37\u00b0C. Once the adapter is ligated to the DNA sequence<\/span>, temperature is a factor of less concern, though care should be taken not to denature the library prior to the agarose gel electrophoresis<\/span> process. Single\u2010stranded DNA<\/span> has a different migration rate in the electrophoresis gel.<\/p>\n

DNA Input Recommendations <\/strong><\/h3>\n

Input DNA Quantification<\/strong><\/h4>\n

The success of DNA<\/span> library preparation depends on the accurate quantification<\/span> of the input DNA<\/span>. Generally recommendation is the input DNA<\/span> should be less than 1 \u03bcg.<\/p>\n

Assessing DNA Quantity and Quality <\/strong><\/h4>\n

Spectrophotometric quantification<\/span> of DNA<\/span> molecules is done at 260 nm<\/span>. Sample purity is measured using the ratio of absorbance at 260 nm<\/span> to 280 nm<\/span>. This value should be in the range of 1.8 – 2.0. Presence of RNA and other nucleotides<\/span> can compromise the measurement of both absorbance. Thus DNA<\/span> collection and purity determines the validity of this method.<\/p>\n

Florescent dyes is used for only double stranded DNA<\/span> quantification<\/span>. This method relies on intercalating fluorescent dyes and are less subject to excess nucleic acids. Calibration curves are prepared for the quantification<\/span> and the process is highly prone to pipetting error. Ensure that pipettes are correctly calibrated and are not used at the volume of their performance specifications.<\/p>\n

Gel electrophoresis<\/span> even reveals the condition of DNA<\/span> in a sample. The presence of impurities, such as detergents or proteins<\/span>, causes smearing of DNA<\/span> bands. RNA is visible at the bottom of the gel. A ladder or smear below a band of interest may indicate nicking or other damage to DNA<\/span>.<\/p>\n

Consistency of Results<\/strong><\/h4>\n

Owing to the fact that fluorescent methods only measure double stranded DNA<\/span> at 260 nm<\/span>, it is used as determinations measure for both single and double stranded DNA<\/span>. It can also be applied to find the denaturation state of the DNA<\/span> sample. Sample preparation can begin with a fixed amount of double\u2010stranded DNA<\/span> prior to fragmentation and cleanup, and thereafter, can be measured by 260 nm<\/span> determination. If the DNA<\/span> amount is less than expected, the amount of input DNA<\/span> can be adjusted before proceeding to the end\u2010repair steps. A further validation step can be performed by analyzing an aliquot for the presence of contaminants by electrophoresis or using an automated instrument, such as the Agilent Bioanalyzer.<\/p>\n

Reagents <\/strong><\/h3>\n

Store the components at \u201015\u00b0 to \u201025\u00b0C.<\/p>\n

    \n
  1. T4<\/span> DNA Ligase<\/span> Buffer with 10 mM ATP<\/span> (100 \u03bcl)<\/li>\n
  2. Klenow Enzyme<\/span> (10 \u03bcl), part<\/li>\n
  3. Klenow Buffer (100 \u03bcl), part<\/li>\n
  4. DNA Ligase<\/span> Buffer 2X (250 \u03bcl)<\/li>\n
  5. Phusion™ DNA<\/span> Polymerase<\/span> (Finnzymes Oy) (250 \u03bcl)<\/li>\n
  6. 10 mM dNTP Mix (20 \u03bcl)<\/li>\n
  7. T4<\/span> PNK (50 \u03bcl)<\/li>\n
  8. 1 mM dATP (100 \u03bcl)<\/li>\n
  9. PE Adapter Oligo Mix (100 \u03bcl)<\/li>\n
  10. PCR<\/span> Primer PE 1.0 (10 \u03bcl)<\/li>\n
  11. T4<\/span> DNA<\/span> Polymerase<\/span> (50 \u03bcl)<\/li>\n
  12. Klenow Exo \u2010 (30 \u03bcl)<\/li>\n
  13. DNA Ligase<\/span> (50 \u03bcl)<\/li>\n
  14. PCR<\/span> Primer PE 2.0 (10 \u03bcl)<\/li>\n<\/ol>\n

    Box 2 <\/strong><\/h3>\n

    Store at Room Temperature<\/p>\n

      \n
    1. Nebulization Buffer (7 ml)<\/li>\n
    2. TE Buffer (10 ml)<\/li>\n
    3. Ultra Pure Water (10 ml)<\/li>\n
    4. Nebulizer Kit (10 each), part # 1000541<\/li>\n<\/ol>\n

      Consumables and Equipment<\/strong><\/h3>\n
        \n
      1. 50X TAE Buffer<\/li>\n
      2. 100 bp Redi\u2010Load DNA<\/span> ladder Invitrogen<\/li>\n
      3. Certified low range ultra agarose<\/li>\n
      4. Compressed air source of at least 32 psi (Do not use CO2 which could alter the pH<\/span> of the nebulizer buffer)<\/li>\n
      5. Disposable scalpels<\/li>\n
      6. Distilled water<\/li>\n
      7. Ethidium Bromide<\/li>\n
      8. Loading buffer (50 mM Tris pH<\/span> 8.0, 40 mM EDTA, 40% (w\/v) sucrose)<\/li>\n
      9. MinElute PCR<\/span> Purification Kit<\/li>\n
      10. Purified DNA<\/span> (1\u20135 \u03bcg, 5 \u03bcg recommended) DNA<\/span> should be as intact as possible, with an OD260\/280 ratio of 1.8\u20132.0<\/li>\n
      11. PVC tubing Intersurgical<\/li>\n
      12. QIAquick Gel Extraction Kit (for 50 samples) or QIAquick Gel Extraction Kit (for 250 samples)<\/li>\n
      13. QIAquick PCR<\/span> Purification Kit<\/li>\n
      14. Benchtop microcentrifuge<\/li>\n
      15. Benchtop centrifuge with swing\u2010out rotor (e.g., Sorvall Legend RT)<\/li>\n
      16. Dark Reader transilluminator or a UV transilluminator<\/li>\n
      17. Electrophoresis unit<\/li>\n
      18. Gel trays and tank<\/li>\n
      19. Thermal cycler<\/li>\n<\/ol>\n

        Fragment DNA<\/strong><\/h3>\n

        Nebulization technique is used for DNA<\/span> fragmentation. This breaks up DNA<\/span> into pieces less than 800 bp in minutes using a disposable device. The end product generated will be double\u2010stranded DNA<\/span> fragments containing 3\u02b9 or 5\u02b9 overhangs.<\/p>\n

        DNA<\/span> is forced through a narrow orifice using a Compressed air source. The solution follows a cyclical route from a collection chamber via a siphon tube back to the sample reservoir and through the orifice again. After 5\u20136 minutes of repeated passing through the orifice, the sample is fragmented to its final size. Nebulization is a very reproducible process and produces random fragments that approximate a Poisson distribution when sequenced.<\/p>\n

        Consumables <\/strong><\/h3>\n
          \n
        1. Nebulizers (box of 10 nebulizers and vinyl accessory tubes)<\/li>\n
        2. Nebulization Buffer (7 ml)<\/li>\n
        3. TE Buffer<\/li>\n
        4. QIAquick PCR<\/span> Purification Kit (QIAGEN, part # 28104)<\/li>\n
        5. Purified DNA<\/span> (1\u20135 \u03bcg, 5 \u03bcg recommended). DNA<\/span> should be as intact as possible, with an OD260\/280 ratio of 1.8\u20132.0<\/li>\n
        6. Compressed Air of at least 32 psi. Do not use CO2 which could alter the pH<\/span> of the nebulizer buffer<\/li>\n
        7. PVC tubing Dimensions: 1\/4 inch ID, 3\/8 inch OD, 1\/16 inch wall, 1 meter length<\/li>\n<\/ol>\n

          Procedure<\/strong><\/h3>\n

          The DNA<\/span> sample to be processed should be highly pure, having an OD 260\/280 ratio of between 1.8 and 2.0, and should be as intact as possible.<\/p>\n

            \n
          1. Remove a nebulizer from the plastic packaging and unscrew the blue lid.<\/li>\n
          2. Using gloves, remove a piece of vinyl tubing from the packaging and slip it over the central atomizer tube. Push it all the way to the inner surface of the blue lid.<\/li>\n
          3. Add 1\u20135 g of Purified DNA<\/span> in a total volume of 50 \u03bcl of TE Buffer to the nebulizer.<\/li>\n
          4. Add 700 \u03bcl Nebulization Buffer to the DNA<\/span> and mix well.<\/li>\n
          5. Screw the lid back on (finger\u2010tight).<\/li>\n
          6. Chill the nebulizer containing the DNA<\/span> solution on ice while performing the next step.<\/li>\n
          7. Connect the compressed air source to the inlet port on the top of the nebulizer with the PVC tubing, ensuring a tight fit.<\/li>\n
          8. Bury the nebulizer in an ice bucket and place it in a fume hood.<\/li>\n
          9. Use the regulator on the compressed air source to ensure the air is delivered at 32\u201035 psi.<\/li>\n
          10. Nebulize for 6 minutes. You may notice vapor rising from the nebulizer; this is normal. Also, the Nebulization Buffer may turn white or appear frozen.<\/li>\n
          11. Centrifuge the nebulizer at 450 x for 2 minutes to collect the droplets from the side of the nebulizer. If necessary, use an old nebulizer as a counter\u2010balance.<\/li>\n
          12. If a centrifuge is not available, then use 2 ml of the binding buffer (PB or PBI buffer) from the QIAquick PCR<\/span> Purification Kit to rinse the sides of the nebulizer and collect the DNA<\/span> solution at the base of the nebulizer.<\/li>\n
          13. Measure the recovered volume. Typically, you should recover 400\u2013600 \u03bcl.<\/li>\n
          14. Follow the instructions in the QIAquick PCR<\/span> Purification Kit to purify the sample solution and concentrate it on one QIAquick column, eluting in 30 \u03bcl of QIAGEN EB.<\/li>\n
          15. Perform a quality control step on the recovered DNA<\/span> to ensure the presence of sufficient DNA<\/span> (i.e., quantify the DNA<\/span> by a 260 nm<\/span> reading, or by a PicoGreen assay, bioanalyzer, or equivalent). Ensure that there is at least 0.5 \u03bcg of DNA<\/span>. If not, then fragment more DNA<\/span>.<\/li>\n<\/ol>\n

            Alternative Fragmentation Methods <\/strong><\/h3>\n

            Sonication <\/strong><\/p>\n

            Sonication is sometimes used as an alternative to nebulization, since sonicators are relatively common in molecular biology<\/span> labs. The temperature of the DNA<\/span> solution should not be raised above ~20\u00b0C. Heating small fragments with high AT content may result in complete denaturation. These single stranded denatured fragments will not ligate to the adapters which are all double stranded. This may lead<\/span> to the under\u2010representation of DNA<\/span> in the final library. Sonication fragments DNA<\/span> in a decreasing but broad range of fragment sizes. Therefore, sonication settings must be chosen carefully to reproducibly generate a majority of fragments in the desired size range.<\/p>\n

            A probe\u2010based sonicator wherein the probe tip is directly immersed into the DNA<\/span> sample may be used, although problems with sample heating, vaporization, and contamination may arise. Alternatively, a cup horn sonicator protects the DNA<\/span> solution DNA<\/span> solution which remain sealed during fragmentation.<\/p>\n

            Hydroshear <\/strong><\/h4>\n

            Hydroshearing fragments DNA<\/span> by a similar mechanism to nebulization. A syringe pump is used to pass the DNA<\/span> solution back and forth through a narrow orifice. As the liquid streams through the orifice, it becomes laminar (i.e., the center of the stream moves faster than the stream closer to the walls of the orifice) and DNA<\/span> molecules are stretched and snapped at the midpoint of their length. Hydroshearing produces fragments in a range of 1,000\u201040,000 bp. It is incapable of generating fragments <1,000 bp in length.<\/p>\n

            Perform End Repair<\/strong><\/h4>\n

            DNA<\/span> fragmentation by physical methods produces heterogeneous ends. These fragments comprise of a mixture of 3\u2019 overhangs, 5\u2019 overhangs, and blunt ends. The overhangs will be of varying lengths and ends may or may not be phosphorylated. End repair is required to transfer these overhangs into blunt ends. The process uses T4<\/span> DNA<\/span> polymerase<\/span> and Klenow enzyme<\/span>. The 3\u02b9 to 5\u02b9 exonuclease activity of these enzymes removes 3\u02b9 overhangs and the polymerase<\/span> activity fills in the 5\u02b9 overhangs. In addition, T4<\/span> PNK in this reaction phosphorylates the 5\u02b9 ends of the DNA<\/span> fragments<\/p>\n

            \u00a0Consumables <\/strong><\/h3>\n
              \n
            1. T4<\/span> DNA Ligase<\/span> Buffer with 10 mM ATP<\/span><\/li>\n
            2. 10 mM dNTP Mix<\/li>\n
            3. T4<\/span> DNA<\/span> Polymerase<\/span><\/li>\n
            4. Klenow Enzyme<\/span><\/li>\n
            5. T4<\/span> PNK<\/li>\n
            6. Water<\/li>\n<\/ol>\n

              QIAquick PCR Purification Kit<\/strong><\/h3>\n
                \n
              1. Prepare the reaction mix on ice in the following order:<\/li>\n<\/ol>\n

                Water 45 \u03bcl, DNA<\/span> Sample 30 \u03bcl, T4<\/span> DNA Ligase<\/span> Buffer with 10 mM ATP<\/span> 10 \u03bcl, 10 mM dNTP Mix 4 \u03bcl, T4<\/span> DNA<\/span> Polymerase<\/span> 5 \u03bcl, Klenow Enzyme<\/span> 1 \u03bcl,\u00a0 T4<\/span> PNK 5 \u03bcl.<\/p>\n

                  \n
                1. Mix gently, but thoroughly, and centrifuge briefly<\/li>\n
                2. Incubate on the thermal cycler for 30 minutes at 20\u00baC.<\/li>\n
                3. Follow the instructions in the QIAquick PCR<\/span> Purification Kit to purify on one QIAquick column, eluting in 32 \u03bcl of QIAGEN EB.<\/li>\n<\/ol>\n

                  Adenylate 3′ Ends <\/strong><\/h3>\n

                  During the adapter ligation reaction there is a possibility of ends of the blunt fragments may ligate with one another. A single \u2018A\u2019 nucleotide is added to the 3\u2019 ends of the blunt fragments to prevent them from ligating to one another.\u00a0\u00a0 A corresponding single \u2018T\u2019 nucleotide on the 3\u2019 end of the adapter provides a complementary overhang for ligating the adapter to the fragment. This strategy ensures a low rate of chimera (concatenated template) formation.<\/p>\n

                  Illumina\u2010Supplied Consumables <\/strong><\/h3>\n
                    \n
                  1. Klenow Buffer<\/li>\n
                  2. 1 mM dATP<\/li>\n
                  3. Klenow Exo \u2010<\/li>\n<\/ol>\n

                    MinElute PCR Purification Kit <\/strong><\/h3>\n

                    Procedure <\/strong><\/h4>\n
                      \n
                    1. Prepare the reaction mix on ice in the following order:<\/li>\n<\/ol>\n

                      DNA<\/span> Sample 32 \u03bcl, Klenow Buffer 5 \u03bcl, 1 mM dATP 10 \u03bcl, Klenow Exo \u2010 3\u00a0 \u03bcl<\/p>\n

                        \n
                      1. Incubate on the thermal cycler for 30 minutes at 37\u00b0C.<\/li>\n
                      2. Follow the instructions in the MinElute PCR<\/span> Purification Kit to purify on one QIAquick MinElute column, eluting in 10 \u03bcl of QIAGEN EB.<\/li>\n<\/ol>\n

                        Ligate Adapters<\/strong><\/h4>\n

                        This process ligates adapters to the ends of the DNA<\/span> fragments. The reaction adds distinct sequences to the 5\u2019 and 3\u2019 ends of each strand in the genomic fragment. Later in the workflow, additional sequences are added by tailed primers during PCR<\/span>. These additional sequences are necessary for library amplification on the flow cell during cluster formation.<\/p>\n

                        Consumables <\/strong><\/h4>\n
                          \n
                        1. DNA Ligase<\/span> Buffer, 2X<\/li>\n
                        2. PE Adapter Oligo Mix<\/li>\n
                        3. DNA Ligase<\/span><\/li>\n
                        4. QIAquick PCR<\/span> Purification Kit<\/li>\n<\/ol>\n

                          \u00a0Procedure <\/strong><\/h4>\n

                          High molar ratio of adapter to fragments increases the yields of adapter ligation. If the ratio is too great, then the yield of adapter dimers also increases.\u00a0 For example, if 1 \u03bcg or less of input DNA<\/span> is used instead of 5 \u03bcg. The volume of adapter oligo mix added in the following procedure is recommended for an initial input DNA<\/span> quantity of 5 \u03bcg (approximately 2.5 \u03bcg post\u2010nebulization or 4\u20135 \u03bcg following covaris shearing). If 1 \u03bcg of input DNA<\/span> is used, then the amount of adapter added to the reaction should be decreased to 5 \u03bcl.<\/p>\n

                            \n
                          1. Prepare the reaction mix on ice in the following order: DNA<\/span> Sample 10 \u03bcl, DNA Ligase<\/span> Buffer 2X 25 \u03bcl, PE Adapter Oligo Mix 10 \u03bcl, DNA Ligase<\/span> 5 \u03bcl.<\/li>\n
                          2. Incubate on the thermal cycler for 15 minutes at 20\u00b0C.<\/li>\n
                          3. Follow the instructions in the QIAquick PCR<\/span> Purification Kit to purify on one QIAquick column, eluting in 30 \u03bcl of QIAGEN EB.<\/li>\n<\/ol>\n

                            Purify Ligation Products <\/strong><\/h4>\n

                            Unligated adapters, or any adapters that may have ligated to one another, is purified and removed. This procedure selects a size\u2010range of sequencing library appropriate for cluster generation.<\/p>\n

                            Size Selection <\/strong><\/h4>\n

                            Gel electrophoresis<\/span> is done for band excision after adapter ligation. Electrophoresis is odne to remove excess adapter and adapter dimers and to tighten the range of fragment sizes. Ligation reaction products are separated on an agarose gel and a ~2 mm wide gel slice containing DNA<\/span> of the desired size is excised.<\/p>\n

                            Excision Range<\/strong><\/h4>\n

                            A 200 bp insert size target (+\/\u2010 1 standard deviation of 20 bp, i.e, a 10% variance) for read lengths of 2\u00d775 bp or shorter. In practice, this translates to a 2 mm gel slice at ~300 bp to account for the length of the adapter sequences flanking the inserts. For read lengths of 2\u00d7100 bp or longer, an insert size range of 300 bp or greater should be targeted for excision from the gel, unless you intentionally want to sequence overlapping read pairs.<\/p>\n

                            Consumables <\/strong><\/h4>\n
                              \n
                            1. Certified Low Range Ultra Agarose<\/li>\n
                            2. 50x TAE Buffer<\/li>\n
                            3. Distilled Water<\/li>\n
                            4. Ethidium Bromide<\/li>\n
                            5. Loading Buffer<\/li>\n
                            6. 100 bp Redi\u2010Load DNA<\/span> Ladder<\/li>\n
                            7. QIAquick Gel Extraction Kit<\/li>\n<\/ol>\n

                              Procedure <\/strong><\/h4>\n
                                \n
                              1. Prepare a 150 ml, 2% agarose gel with distilled water and TAE according to the manufacturer\u2019s instructions. The final concentration of TAE should be 1X.<\/li>\n
                              2. Add Ethidium Bromide (EtBr) after the TAE\u2010agarose has cooled. The final concentration of EtBr should be 400 ng\/ml (i.e., add 60 \u03bcg EtBr to 150 ml of 1X TAE\u2010agarose).<\/li>\n
                              3. Cast the gel using a comb that can accommodate 56 \u03bcl in each well. Recommended well size: 1 mm (length) x 8 mm (width) x 7 mm (height).<\/li>\n
                              4. Add 3 \u03bcl of Loading Buffer to 8 \u03bcl of the 100 bp Redi\u2010Load Ladder.<\/li>\n
                              5. Add 10 \u03bcl of Loading Buffer to 30 \u03bcl of the DNA<\/span> from the purified ligation reaction.<\/li>\n
                              6. Load 10 \u03bcl of the ladder onto one lane of the gel<\/li>\n
                              7. Load the entire sample onto another lane of the gel, leaving a gap of at least one empty lane between ladder and sample.<\/li>\n
                              8. Run gel at 120 V for 60 minutes (6 V\/cm).<\/li>\n
                              9. View the gel on a Dark Reader transilluminator or a UV transilluminator.<\/li>\n
                              10. Place a clean scalpel vertically above the sample in the gel at the desired size of the template.<\/li>\n
                              11. Excise a 2 mm slice of the sample lane at approximately 400 bp using the markers<\/span> as a guide.<\/li>\n
                              12. Discard the scalpel to avoid sample cross\u2010contamination.<\/li>\n
                              13. Follow the instructions in the QIAquick Gel Extraction Kit to purify on one QIAquick column, eluting in 30 \u03bcl of QIAGEN EB.<\/li>\n<\/ol>\n

                                Enrich DNA Fragments <\/strong><\/h3>\n

                                PCR<\/span> is used to selectively enrich those DNA<\/span> fragments that have adapter molecules on both ends and to amplify the amount of DNA<\/span> in the library for accurate quantification<\/span>. The PCR<\/span> is performed with two primers that anneal to the ends of the adapters.<\/p>\n

                                PCR Amplification <\/strong><\/h3>\n

                                The PCR<\/span> amplification step of the protocol performs four key functions:<\/p>\n

                                  \n
                                1. Add Sequences<\/li>\n<\/ol>\n

                                  Additional sequences are added to the ends of the adapters for cluster generation.<\/p>\n

                                    \n
                                  1. Enrich Fragments<\/li>\n<\/ol>\n

                                    PCR<\/span> enriches for fragments that have adapters ligated on both ends.<\/p>\n

                                      \n
                                    1. Enrich Templates<\/li>\n<\/ol>\n

                                      PCR<\/span> enriches for templates that include the non\u2010template \u2018A\u2019 nucleotide added during the A\u2010tailing step of the protocol and therefore eliminates adapter dimers.<\/p>\n

                                        \n
                                      1. Provides Material<\/li>\n<\/ol>\n

                                        PCR<\/span> provides enough material to enable reliable quantification<\/span> of the final library if spectrophotometric or fluorometric methods are used.<\/p>\n

                                        Consumables<\/strong><\/h3>\n
                                          \n
                                        1. Phusion DNA<\/span> Polymerase<\/span> (Finnzymes Oy)<\/li>\n
                                        2. PCR<\/span> Primer PE 1.0<\/li>\n
                                        3. PCR<\/span> Primer PE 2.0<\/li>\n
                                        4. Ultra Pure Water<\/li>\n
                                        5. QIAquick PCR<\/span> Purification Kit<\/li>\n<\/ol>\n

                                          Procedure <\/strong><\/h3>\n

                                          If the initial input DNA<\/span> is 1 \u03bcg or less, then more ligated fragments should be added to the PCR<\/span> reaction. If you are using 0.5 \u03bcg, adjust the process as described in the following ratio of Input of DNA<\/span> to Library Prep :Volume of Purified Library into PCR:Volume of Water:Number of PCR<\/span> Cycles case i) 5 \u03bcg:1 \u03bcl:22 \u03bcl:10 or 0.5 \u03bcg: 10 \u03bcl:13 \u03bcl:12<\/p>\n

                                            \n
                                          1. Prepare the reaction mix on ice in a 200 \u03bcl thin wall PCR<\/span> tube in the following order: DNA<\/span> x \u03bcl, Ultra Pure Water to increase DNA<\/span> volume to 23 \u03bcl, PCR<\/span> primer PE 2.0 1 \u03bcl, PCR<\/span> primer PE 1.0 1 \u03bcl, Phusion DNA<\/span> Polymerase<\/span> (Finnzymes Oy) 25 \u03bcl.<\/li>\n
                                          2. Mix gently, but thoroughly, and centrifuge briefly.<\/li>\n
                                          3. Amplify using the following PCR<\/span> process:<\/li>\n
                                          4. 30 seconds at 98\u00b0C<\/li>\n
                                          5. 10 or 12 cycles of:<\/li>\n<\/ol>\n

                                            40 seconds at 98\u00b0C<\/p>\n

                                            30 seconds at 65\u00b0C<\/p>\n

                                            30 seconds at 72\u00b0C<\/p>\n

                                              \n
                                            1. 5 minutes at 72\u00b0C<\/li>\n
                                            2. Hold at 4\u00b0C<\/li>\n
                                            3. Follow the instructions in the QIAquick PCR<\/span> Purification Kit to purify on one QIAquick column, eluting in 30 \u03bcl of QIAGEN EB.<\/li>\n<\/ol>\n

                                              Purify Final Product <\/strong><\/h3>\n

                                              This process further purifies the products of the PCR<\/span> ligation reaction on a gel to select a size\u2010range of templates appropriate for subsequent cluster generation.<\/p>\n

                                              \u00a0Consumables <\/strong><\/h3>\n
                                                \n
                                              1. Certified Low Range Ultra Agarose<\/li>\n
                                              2. 50x TAE Buffer<\/li>\n
                                              3. Distilled Water<\/li>\n
                                              4. Ethidium Bromide<\/li>\n
                                              5. Loading Buffer<\/li>\n
                                              6. 100 bp Redi\u2010Load DNA<\/span> ladder<\/li>\n
                                              7. QIAquick Gel Extraction Kit<\/li>\n<\/ol>\n

                                                Procedure <\/strong><\/h4>\n
                                                  \n
                                                1. Prepare a 150 ml, 2% agarose gel with distilled water and TAE according to the manufacturer\u2019s instructions. The final concentration of TAE should be 1X.<\/li>\n
                                                2. Add EtBr after the TAE\u2010agarose has cooled. The final concentration of EtBr should be 400 ng\/ml (i.e., add 60 \u03bcg EtBr to 150 ml of 1x TAE\u2010agarose).<\/li>\n
                                                3. Cast the gel using a comb that can accommodate 56 \u03bcl in each well. Recommended well size: 1 mm (length) x 8 mm (width) x 7 mm (height).<\/li>\n
                                                4. Add 3 \u03bcl of Loading Buffer to 8 \u03bcl of the ladder.<\/li>\n
                                                5. Add 10 \u03bcl of Loading Buffer to 30 \u03bcl of the DNA<\/span> from the purified PCR<\/span> reaction.<\/li>\n
                                                6. Load 10 \u03bcl of the 100 bp Redi\u2010Load ladder onto one lane of the gel.<\/li>\n
                                                7. Load the entire sample onto another lane of the gel, leaving a gap of at least one empty lane between ladder and sample.<\/li>\n
                                                8. Run gel at 120 V for 60 minutes (6 V\/cm).<\/li>\n
                                                9. View the gel on a Dark Reader transilluminator or a UV transilluminator.<\/li>\n
                                                10. Place a clean scalpel vertically above the sample in the gel at the desired size of the template.<\/li>\n
                                                11. Excise a 2 mm slice of the sample lane at approximately 400 bp using the markers<\/span> as a guide<\/li>\n
                                                12. Discard the scalpel.<\/li>\n
                                                13. Follow the instructions in the QIAquick Gel Extraction Kit to purify on one QIAquick column, eluting in 30 \u03bcl of QIAGEN EB.<\/li>\n<\/ol>\n

                                                  Validate Library<\/strong><\/h3>\n
                                                    \n
                                                  1. Check the template size distribution by running an aliquot of the library on a gel or an Agilent Bioanalyzer.<\/li>\n
                                                  2. If validating by gel, load 10% of the volume of the library on a gel and check that the size range is as expected: a narrow smear similar in size to the DNA<\/span> excised from the gel after the ligation.<\/li>\n
                                                  3. If using Agilent Bioanalyzer, load 1 \u03bcl of the library on Agilent chip.<\/li>\n<\/ol>\n

                                                    Library Quantification<\/strong><\/h3>\n

                                                    As described for sample input quantification<\/span>, any method of measuring DNA<\/span> concentration has certain advantages and potential drawbacks. Optical density, Bioanalyzer and qPCR can be used for quantification<\/span>.<\/p>\n","protected":false},"excerpt":{"rendered":"

                                                    Introduction This is a protocol for preparing genomic library for paired end sequencing. Sequencing library are produced by adding adapter sequences onto the ends of the DNA fragments. These chemically synthesized single stranded or double stranded oligonucleotide can be joined to other DNA or RNA molecules. Sample Prep Workflow The input genomic DNA is first […]<\/p>\n","protected":false},"author":2,"featured_media":0,"parent":401,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":{"_et_pb_use_builder":"","_et_pb_old_content":"","_et_gb_content_width":"","footnotes":""},"class_list":["post-1606","page","type-page","status-publish","hentry"],"_links":{"self":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1606","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/users\/2"}],"replies":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/comments?post=1606"}],"version-history":[{"count":0,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/1606\/revisions"}],"up":[{"embeddable":true,"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/pages\/401"}],"wp:attachment":[{"href":"https:\/\/www.mybiosource.com\/learn\/wp-json\/wp\/v2\/media?parent=1606"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}