This kit is recommended for users who

• have a low starting amount of DNA
• want to optimise their sequencing experiment for throughput
• require control over read length
• are interested in targeted amplicon sequencing

The PCR Sequencing Kit is designed to prepare sequencing libraries when there is a limited amount of starting gDNA available.

The gDNA is fragmented in a Covaris g-TUBE. The sheared ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then adapters which contain primer binding sites are ligated onto the prepared ends. An amplification step follows using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

The kit involves ~50 mins pre-PCR work. The PCR step is variable (depends on the number of cycles, polymerase speed and template length). The standard protocol suggests starting with 100 ng of gDNA: less can be used, but it may be necessary to increase the number of PCR cycles. The post-PCR work is about 10 mins.

 The PCR Sequencing Kit features:

The fragment length produced after PCR is dependent on the length of the material input into the PCR and is limited by the processivity of the DNA polymerase. This means that superior fragment lengths can be achieved with the PCR Sequencing Kit, compared to the Rapid PCR Barcoding Kit, which is limited to ~2 kb. Therefore this kit offers a solution to those who have limited amounts of starting material (i.e. need to do PCR) but require longer reads than are offered by the Rapid PCR Barcoding Kit.

Typical read-length histograms observed when preparing libraries with the PCR Sequencing Kit (A) and the Rapid PCR Barcoding Kit (B).

This kit also offers a method whereby users are able to tag their own specific amplicons with the same 5’ group, simplifying downstream post-PCR processing. This is done via a four-primer PCR. Users add a 5’ tail to their own primer sequences and this acts a priming site for a set of generic “outer” primers (supplied in the PCR Sequencing Kit) and these primers contain the 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

Further considerations:

As well as being used to generate more material (where starting material is limiting), PCR can be used where sample purity is compromised (which can inhibit library preparation efficiency); PCR selects for molecules which have been successfully adapted, and generate more of them. The original impurity is lost or diluted. It is often observed that a sample which produces poor sequencing results can be improved by inclusion of PCR. In instances where less than 1 ng of input material is available, we recommend whole genome amplification.

A PCR Barcoding Kit is available for multiplexing up to 12 samples.

Equipment and consumables for PCR Sequencing Kit

The PCR Sequencing Kit is designed to prepare sequencing libraries when there is a limited amount of starting gDNA available.

The gDNA is fragmented in a Covaris g-TUBE. The sheared ends are repaired and dA-tailed using the NEBNext End Repair/dA-tailing module, and then adapters which contain primer binding sites are ligated onto the prepared ends. An amplification step follows using primers that contain 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

The kit involves ~50 mins pre-PCR work. The PCR step is variable (depends on the number of cycles, polymerase speed and template length). The standard protocol suggests starting with 100 ng of gDNA: less can be used, but it may be necessary to increase the number of PCR cycles. The post-PCR work is about 10 mins.

This kit also offers a method whereby users are able to tag their own specific amplicons with the same 5’ group, simplifying downstream post-PCR processing. This is done via a four-primer PCR. Users add a 5’ tail to their own primer sequences and this acts a priming site for a set of generic “outer” primers (supplied in the PCR Sequencing Kit) and these primers contain the 5’ tags which facilitate the ligase-free attachment of Rapid Sequencing Adapters.

Example experiment using the PCR Sequencing Kit (SQK-PSK004) - for illustration purposes only

Safety Data Sheet for PCR Sequencing Kit

Safety Data Sheet for Flow Cell Priming Kit

The PCR Sequencing Kit contains sufficient reagents to generate 6 sequencing ibraries.

The Flow Cell Priming Kit Expansion Pack is also supplied.

Multiplexing with barcoding for up to 12 samples is available with an alternative purchase: PCR Barcoding Kit (SQK-PBK004).

Consumables

• Agencourt AMPure XP beads
• NEBNext End repair / dA-tailing Module (E7546)
• NEB Blunt/TA Ligase Master Mix (M0367)
• 1.5 ml Eppendorf DNA LoBind tubes
• 0.2 ml thin-walled PCR tubes
• Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
• Freshly prepared 70% ethanol in nuclease-free water
• LongAmp Taq 2X Master Mix (e.g. NEB M0287)
• 10 mM Tris-HCl pH 8.5 with 50 mM NaCl

Optional consumables

• Covaris g-TUBE
• Exonuclease I (NEB, M0293)

Equipment

• Hula mixer (gentle rotator mixer)
• Magnetic rack
• Microfuge
• Vortex mixer
• Ice bucket with ice
• Timer
• Thermal cycler
• P1000 pipette and tips
• P100 pipette and tips
• P20 pipette and tips
• P10 pipette and tips
• P2 pipette and tips

Optional Equipment

• Agilent Bioanalyzer (or equivalent)
• Qubit fluorometer (or equivalent for QC check)
• Eppendorf 5424 centrifuge (or equivalent)

The PCR Sequencing Kit can be used together with:

Kits

• Flow Cell Wash Kit (EXP-WSH003)

Flow cells

• FLO-MINSP6
• FLO-MIN106D

Devices

• MinION Mk 1B
• GridION X5