SOLiD4 barcoded fragment library preparation and sequencing

CHAPTER 7: MUTATION SCREENING APPROACH 2: NGS SOLiD SEQUENCING OF

7.2.3 Preparation of LR-PCR products for next generation sequencing

7.2.3.2 SOLiD4 barcoded fragment library preparation and sequencing

Sequencing libraries were prepared by the service provider (Life Technologies, Melbourne, Australia) as the first step in which samples (amplicons in two 96-well plates) were adapted for sequencing by oligonucleotide ligation and detection (SOLiD, Life Technologies) sequencing. The samples comprised of fourteen amplicons from each of the eight sheep which were pooled in

equimolar ratios into two pools of seven non-overlapping amplicons (Table 7.4). Each of this pool represents a library, which means there are 2 libraries for each animal.

The barcoded fragment library was utilised in this study. Each library is tagged with a barcode, containing unique 5 - 10 base sequences, on one of the adaptors, to enable multiplexed sequencing analysis where multiple samples are run simultaneously in a single sequencing run.

Preparation of the barcoded fragment libraries using the amplicon pools was performed using the SOLiD4 fragment library barcoding kit (Applied Biosystem, USA) and the SOLiD fragment library construction kit (Applied Biosystem, USA). A summary of the workflow description of the preparation of barcoded fragment libraries and a schematic diagram of a typical barcoded fragment are shown below in Figures 7.2 and 7.3, respectively.

Figure 7.2: Preparation workflow for the barcoded fragment libraries (adapted from Applied Biosystems SOLiD4 system library preparation guide, April 2010).

Summary of construction of the barcoded fragment libraries are as follows:

1. Each pool of seven amplicons was sheared into small DNA fragments with a mean fragment size of 165 bp and a fragment size range of 150 to 180 bp, using the Covaris S2 System.

2. DNA ends of the sheared DNA fragments were end-repaired with End Polishing Enzymes 1 and 2 and purified with the PureLink PCR purification kit (Applied Biosystems, USA).

3. Ends of the end-repaired DNA fragments in each pool of fragmented amplicons were ligated with adaptors (Multiplex P1 Adaptor and a Multiplex P2 Adaptor) and purified with the PureLink purification kit (Applied Biosystems, USA). The Multiplex P2 adaptors included unique barcode sequences (total of 16 barcodes with 1 barcode for each of the two pools of seven amplicons for each of the eight sheep) with each barcode subsequently generating a single fragment library to be multiplexed in the same sequencing run. Sequences for Multiplex P1 and P2 adaptors used for barcoded fragment library construction are listed in Table 7.3. An example of the pool-barcode arrangement is as follows: For animal CPW 156, barcode 1 was ligated to pool 1 (amplicons 1,3,5,7,9,11,13) and barcode 9 was ligated to pool 2 (amplicons 2,4,6,8,10,12,14). Barcode labelings of the remaining seven animals are shown in Table 7.4.

4. After adaptor-ligation and purification, DNA underwent nick translation and was amplified for nine cycles using primers specific to the Multiplex P1 and Multiplex P2 adaptors (Multiplex library PCR-1: CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT and Multiplex library PCR-2: CTGCCCCGGGTTCCTCATTCT primers). After amplification, the library was purified with a PureLinkPCR purification kit (Applied Biosystems, USA). 5. The library was quantitated using the SOLiD Library TaqMan quantitation kit (PN

4449639), according to the manufacturers instructions described in ‘SOLiD4 system library quantitation with the SOLiD library TaqMan quantitation kit’.

6. The barcoded fragment libraries were pooled in equal molar amounts.

Once the barcoded fragment libraries were constructed, the templated bead preparation was performed following the manufacturer’s instructions (Applied Biosystems SOLiD system templated bead preparation guide, March 2010). In summary, this involved clonal amplification using an emulsion PCR of each library template on the SOLiD P1 DNA beads, enrichment of the templated beads and deposition of the beads onto a sequencing slide. The beads were then sequenced on the SOLiD Analyzer on a single segment of a four-quadrant slide, with a 50 bp read length expected for the SOLiD4 system. An overview of the sequencing chemistry for the templated bead preparation and sequencing is described at: http://www.appliedbiosystems.com/absite/us/en/home/applications-technologies/SOLiD-next-

generation-sequencing/next-generation-systems/SOLiD-sequencing-chemistry.html.

Figure 7.3: Schematic diagram of a typical SOLiD4™ barcoded fragment after library construction (adapted from Applied Biosystems SOLi4 system library preparation guide, April 2010).

Table 7.3: Multiplex P1 and P2 adaptors used for barcoded fragment library construction. Each adaptor consists of two sequences to form a double strand as shown in Fig 7.1. The bold sequence flanked by the internal adaptor sequence on the 5’ end and the P2 adaptor sequence on the 3’ end is the barcode sequence

Adaptor and barcode Sequence 5'>3' Length

Multiplex P1 Adaptor ATCACCGACTGCCCATAGAGAGGTT 25

CCTCTCTATGGGCAGTCGGTGAT 23

Multiplex P2 Adaptor with Barcode- 001

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGTGTAAGAGGCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

002

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTAGGGAGTGGTCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

003

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTATAGGTTATACTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

004

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGGATGCGGTCCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

005

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGTGGTGTAAGCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

006

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGCGAGGGACACTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

007

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGGGTTATGCCCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

008

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGAGCGAGGATCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

009

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTAGGTTGCGACCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

010

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGCGGTAAGCTCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

011

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGTGCGACACGCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

012

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTAAGAGGAAAACTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

013

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGCGGTAAGGCCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

014

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGTGCGGCAGACTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

015

CGCCTTGGCCGTACAGCAG 19

CTGCCCCGGGTTCCTCATTCTCTGAGTTGAATGCTGCTGTACGGCCAAGGCG 52 Multiplex P2 Adaptor with Barcode-

016

CGCCTTGGCCGTACAGCAG 19

Table 7.4: Amplicon pool-barcode arrangements used in the SOLiD barcoded fragment library preparations. Each box represents an individual sheep and the two-barcoded pools of seven equimolar non-overlapping amplicons.

CPW156 (SH_N) SH1038/08 (SH_C2) Barcode-001 (sheep1_1)* Barcode-009 (sheep1_9)* Barcode-005 (sheep5_5)* Barcode-013 (sheep5_13)* 1ar, 2ir, 3dr, 4ar, 5ir, C1cr,

C3cr