Palindromic forked adapters

Library construction was performed as described in (Fisher et al., 2011 (link)), with the following modifications: initial genomic DNA input into shearing was reduced from 3μg to 20–250ng in 50μL of solution. For adapter ligation, Illumina paired-end adapters were replaced with palindromic forked adapters, purchased from Integrated DNA Technologies (IDT), with unique dual-indexed molecular barcode sequences to facilitate downstream pooling. Kapa HyperPrep reagents in 96-reaction kit format were used for end repair/A-tailing, adapter ligation, and library enrichment PCR. In addition, during the post-enrichment SPRI cleanup, elution volume was reduced to 30μL to maximize library concentration, and a vortexing step was added to maximize the amount of template eluted. After library construction, libraries were pooled into groups of up to 96 samples. Hybridization and capture were performed using the relevant components of Illumina’s Nextera Exome Kit and following the manufacturer’s suggested protocol, with the following exceptions: First, all libraries within a library construction plate were pooled prior to hybridization. Second, the Midi plate from Illumina’s Nextera Exome Kit was replaced with a skirted PCR plate to facilitate automation. All hybridization and capture steps were automated on the Agilent Bravo liquid handling system.

For whole-exome sequencing (WES), the AllPrep DNA/RNA Mini Kit (Qiagen, Hilden, Germany) was used for extraction of genomic DNA. DNA was quantified in triplicate using a standardized PicoGreen^® dsDNA Quantitation Reagent (Invitrogen, Carlsbad, CA) assay. The quality control identification check was performed using fingerprint genotyping of 95 common SNPs by Fluidigm Genotyping (Fluidigm, San Francisco, CA). Library construction was performed using the KAPA Library Prep kit, with palindromic forked adapters from Integrated DNA Technologies. All library construction, hybridization and capture steps were automated on the Agilent Bravo liquid handling system. Flowcells were sequenced utilizing Sequencing-by Synthesis chemistry for HiSeq 4000 flowcells. Each pool of whole exome libraries was sequenced on paired 76 cycle runs with two 8 cycle index reads across the number of lanes needed to meet coverage for all libraries in the pool (raw data available on request).

DNA libraries for massively parallel sequencing were generated as previously described^{32 (link)} with the following modifications: the initial genomic DNA input into the shearing step was reduced from 3 μg to 10–100 ng in 50 μl of solution. For adaptor ligation, Illumina paired-end adapters were replaced with palindromic forked adapters (purchased from Integrated DNA Technologies) with unique 8-base index molecular barcode sequences included in the adaptor sequence to facilitate downstream pooling. With the exception of the palindromic forked adapters, all reagents used for end repair, A-base addition, adaptor ligation, and library enrichment PCR were purchased from KAPA Biosciences in 96-reaction kits. In addition, during the post-enrichment solid phase reversible immobilization bead cleanup, elution volume was reduced to 20 μl to maximize library concentration, and a vortexing step was added to maximize the amount of template eluted from the beads. Libraries with concentrations above 40 ng μl⁻¹, as measured by a PicoGreen assay automated on an Agilent Bravo instrument, were considered acceptable for hybrid selection and sequencing.

DNA libraries for massively parallel sequencing were generated as previously described^{48 (link)} with the following modifications: the initial genomic DNA input into the shearing step was reduced from 3μg to 10-100ng in 50μL of solution. For adapter ligation, Illumina paired-end adapters were replaced with palindromic forked adapters (purchased from Integrated DNA Technologies) with unique dual indexed 8 base index molecular barcode sequences included in the adapter sequence to facilitate downstream pooling. With the exception of the palindromic forked adapters, all reagents used for end repair, A-base addition, adapter ligation, and library enrichment PCR were purchased from KAPA Biosciences in 96-reaction kits. In addition, during the post-enrichment solid phase reversible immobilization (SPRI) bead cleanup, elution volume was reduced to 30μL to maximize library concentration, and a vortexing step was added to maximize the amount of template eluted.

Library construction was performed as described in,^{110 (link)} with the following modifications: initial genomic DNA input into shearing was reduced from 3 μg to 20–250 ng in 50 μL of solution. For adapter ligation, Illumina paired-end adapters were replaced with palindromic forked adapters, purchased from Integrated DNA Technologies, with unique dual-indexed molecular barcode sequences to facilitate downstream pooling. Kapa HyperPrep reagents in 96-reaction kit format were used for end repair/A-tailing, adapter ligation, and library enrichment PCR. In addition, during the post-enrichment SPRI cleanup, elution volume was reduced to 30 μL to maximize library concentration, and a vortexing step was added to maximize the amount of template eluted.

DNA libraries for massively parallel sequencing were generated as previously described (43 (link)) with the following modifications: the initial genomic DNA input into the shearing step was reduced from 3 μg to 10-100 ng in 50 μL of solution. For adapter ligation, Illumina paired-end adapters were replaced with palindromic forked adapters (Integrated DNA Technologies) with unique 8-base index molecular barcode sequences included in the adapter sequence to facilitate downstream pooling. With the exception of the palindromic forked adapters, all reagents used for end repair, A-base addition, adapter ligation, and library enrichment PCR were purchased from KAPA Biosciences in 96-reaction kits. In addition, during the post-enrichment solid phase reversible immobilization (SPRI) bead cleanup, elution volume was reduced to 20 μL to maximize library concentration, and a vortexing step was added to maximize the amount of template eluted from the beads. Libraries with concentrations above 40 ng/μL, as measured by a PicoGreen assay automated on an Agilent Bravo instrument, were considered acceptable for hybrid selection and sequencing.