Whole genome amplified lung DNA was first treated with KAPA Beads (Roche; Basel, Switzerland) to remove any residual buffer or EDTA prior to library preparation. Sequencing libraries were then created using KAPA HyperPLUS reagents (Roche) on the Hamilton NGS Star liquid handler (Hamilton Robotics; Cary, NC, USA) according to the manufacturer’s instructions. Briefly, samples were enzymatically fragmented, end repaired, A-tailed, and ligated to sample barcodes (Integrated DNA Technologies [IDT]; Coralville, IA, USA). Following library preparation and amplification, libraries were normalized and pooled for exome capture. Sequence capture was performed on the Hamilton NGS Star liquid handler using the IDT xGen Exome Research panel according to the rapid capture protocol. Captured libraries were visualized on the AATI Fragment Analyzer (Agilent Technologies; Santa Clara, CA, USA) for quality. Finally, samples were quantitated by qPCR with KAPA Quant (Roche) and normalized prior to pooling and sequencing on the Illumina NovaSeq 6000 sequencer (Illumina, Inc.; San Diego, CA, USA).
Kapa beads
Manufactured by Roche
Sourced in Switzerland
Kapa beads are magnetic beads used for nucleic acid purification and isolation in various molecular biology and genomics applications. They provide a efficient and scalable platform for DNA/RNA extraction and sample preparation.
Automatically generated - may contain errors
Lab products found in correlation
Aati fragment analyzer, by Agilent Technologies (1 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
T4 dna ligase, by Qiagen (1 mentions)
Dynabeads myone streptavidin t1 beads, by Thermo Fisher Scientific (1 mentions)
Phusion dna polymerase, by New England Biolabs (1 mentions)
Kapa rna hyper riboerase hmr kit, by Roche (1 mentions)
Ercc rna spike in mix, by Thermo Fisher Scientific (1 mentions)
Amplicon ez service, by Illumina (1 mentions)
4 protocols using kapa beads
1
Whole Genome Amplification and Exome Sequencing
2
Loop PCR for Hairpin Synthesis
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Loop PCR was performed using a forward primer containing either an isoG base (to stop the polymerase and therefore create a 5’ overhang of known sequence) or a nickase site, and a reverse primer containing a loop sequence (the sequence of the oligonucleotides used can be found in Supplementary Data 1 ). PCR was performed with Phusion DNA polymerase (NEB®) and we used a slow ramp for the elongation step from 72 °C to 98 °C to allow the synthesis of the loop on the reverse primer. After 20 to 40 cycles of PCR, depending on the expression of the target, exonuclease I (ExoI, NEB®) was added to remove the single-stranded DNA bearing a 5’ loop, as well as the remaining primers. Two more cycles were performed to fill in DNA molecules with a 3’ loop, and a second ExoI step was used before the PCR products were purified with columns (Qiagen or NEB®) or Kapa beads (Roche Diagnostics). When isoG forward primer was used, a Y-shape containing an isoC overhang was ligated using T4 DNA ligase (Enzymatics), followed by agarose gel purification. In the case of the nickase site, PCR fragments were treated with Nb.BbvCI nickase (NEB®) before purification and ligating the Y-shape with the correct overhang. The final hairpins were attached to Dynabeads™ MyOne™ streptavidin T1 beads (Invitrogen).
3
RNA-Seq Library Preparation Protocol
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RNAs for RNA-Seq were prepared as described for RT-qPCR. For RNA-Seq library preparation, 1 μg of RNA was used as input with 2 μL of a 1:100 dilution of ERCC RNA Spike-In Mix (ThermoFisher Scientific, catalog 4456740) and the KAPA RNA Hyper + RiboErase HMR kit was used (Roche, catalog 8098131702) following kit instructions for library prep using KAPA beads (Roche, catalog KK8001). Step-by-step details for library preparation are provided in the Supplementary Data file associated with this article.
4
Amplicon generation and sequencing protocol
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Using NEB Q5 hot start polymerase (New England BioLabs, UK), amplicons (500 bp) were generated in 25 μl reactions. Sample volume of 1 μl (~ 2 ng/μl) was used, with an average final primer concentration of 0.5 μM in each PCR. The amplification was conducted as follows: hot-start polymerase activation for 3 min at 95 °C, followed by 30 cycles of 95 °C for 10 s, 60 °C for 30 s and 72 °C for 45 s, followed by a final elongation step of 72 °C for 2 min. Post-multiplex PCR reaction, amplicons were visualised on a 1% agarose gel to confirm band size. Multiplexed PCR amplicons were first pooled by sample, and then with other samples with different 5ʹ barcode combinations. Sample pools were purified using Roche Kapa beads following manufacturer’s instructions. A bead to sample ratio of 0.7:1 was used to remove excess primers and PCR reagents. The Qubit 2.0 fluorimeter HS DNA kit was used to quantify the pool concentration. Illumina adaptors and barcodes were ligated to the sample pool as a part of the Illumina-based Amplicon-EZ service (Genewiz, UK). The indexed pool was then sequenced using a 2 × 250 bp paired end configuration on an Illumina MiSeq. A minimum of 50,000 reads were attained per pool, equating to at least 290 reads per amplicon in a pool of 170 amplicons (at a low cost of < US$0.5 per amplicon).
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