Ampure xp bead

Three RAD libraries with twelve individuals each were prepared following a modified RAD sequencing protocol [1 (link)], using PstI-HF (New England BioLabs) restriction enzyme to digest 300 ng of genomic DNA per sample. Digested DNA was ligated to P1 barcoded adapters using twelve different barcodes for each library. Adapter-ligated fragments were pooled and sheared targeting a 500 bp average fragment size using a sonicator. To remove adapter dimers, libraries were purified with Agencourt AMPure XP (Beckman Coulter) magnetic beads after P2 adapter ligation with a volume DNA/beads ratio of 1:0.8. After end-repair using a commercial kit (New England BioLab), libraries were amplified by Polymerase Chain Reaction (PCR) performing an initial denaturation step at 98°C for 30 s, followed by 18 cycles of one denaturation step at 98°C for 10 s, annealing at 65°C for 30 s, extension at 72°C for 30 s and a final 5 min extension step. PCR-enriched libraries were purified with AMPure XP beads and the DNA concentration of each library was quantified in a Qubit 2.0 (Invitrogen). Libraries, in a proportional representation, were paired end sequenced in three lanes of an Illumina HiSeq 2000 at Genepool (Ashworth Laboratories).

Genomic DNA was extracted from pooled young leaves (20–25 days) of both parents and RILs using the DNeasy plant kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. DNA quality of each sample was checked on 0.8% agarose gel. Furthermore, DNA quantification was done using a Qubit 2.0 fluorometer (Themo Fisher Scientific Inc., Waltham, MA, USA). ddRAD–Seq protocol (modified GBS) was followed in the present study.
To perform GBS, genomic DNA was double digested with SphI and MlucI restriction enzymes (NEB, UK) and fractionated in 2% agarose gel to check the product size. The digested fragments were cleaned (Agencourt AMpure XP beads, Invitrogen, Waltham, MA, USA) using standard protocols. The ligation enzyme, T4 DNA ligase (NEB, England) was used to ligate the unique barcode adapters (4–8 nt sequence) at 16 °C for 30 min and heat-inactivated at 65 °C for 10 min.
This was followed by indexing with the addition of index-1 and index-2 (6–8 nt long) for multiplexing sequencing libraries in NGS Illumina. These libraries were PCR amplified (8–12 cycles) using a Phusion^TM polymerase kit (Fisher Scientific, Loughborough, UK) followed by AMPure bead cleanup for purification to remove excess adapters and were sequenced on the Illumina HiSeqX platform (Illumina Inc, San Diego, CA, USA).

Eukaryotic rRNA was depleted using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat). After rRNA depletion, cDNA was synthesized from residual total RNA by RT-VILO (Invitrogen) reaction following manufacturer’s instructions. Random amplification or the material was then performed with QuantiTect Whole Transcriptome kit (QIAGEN) according to the manufacturer’s protocol. Amplified DNA was then purified using AMPure XP beads and submitted to Qubit quantification using dsDNA BR Assay Kit and Qubit 3.0 fluorimeter (Invitrogen). Complementary to the un-targeted approach, we also used an adapted version of the published protocol from the ARTIC Network (28 ) using ARTIC primer scheme version 3, which produces ~400 bp overlapping amplicons over the SARS-CoV-2 genome.

Array capture was performed via the Agilent SureSelectXT2 Target Enrichment System as previously described [14 (link)]. Briefly, array hybridization was captured by mixing the pooled libraries with a buffer solution and oligo-blockers, which was incubated for 24 h at 65 °C. The hybridized library molecules were performed with Dynabeads® MyOne™ Streptavidin T1 (Invitrogen, #65601). The captured library was amplified as following: 21 μl 2× KAPA HiFi HotStart ReadyMix, 1 μl 5 μM primer, 20 μl captured library beads suspension. PCR amplification program was 98 °C 2 min; 98 °C 30 s; 65 °C 30 s; 72 °C 30 s, 13 cycles and a final step at 72 °C for 4 min. Purifications between procedures were conducted using Agencourt AMPure XP beads and the libraries were evaluated with Qubit dsDNA HS Assay kit (Invitrogen, Q32851). Finally, DNA libraries of the proband were analyzed by whole exome sequencing (WES). WES was carried out on the HiSeq2500 platform as paired-end 200-bp reads. Illumina Sequence Control Software (SCS) was used to evaluate the sequencing data, thus removing adapter sequences in the raw data and discarding low-quality sequencing reads. Conventional Sanger sequencing of the SPAST gene was further performed in 15 individuals from the Chinese family.