Truseq dna sample preparation v2 guide

Amplicon sequencing was carried out on a MiSeq (Illumina, San Diego, USA) in seven runs. All amplicons belonging to a respective individual were pooled in equimolar amounts, fragmented by sonification using a Bioruptor (Diagenode, Denville, USA) and subsequently used for library preparation. The libraries were prepared as recommended by Illumina (TruSeq DNA Sample Preparation v2 Guide). Adaptor-ligated fragments were size selected on a two percent low melt agarose gel to an average insert size of 500 bp. Fragments that carry adaptors on both ends were enriched by PCR. Final libraries were quantified using PicoGreen (Quant-iT, Fisher Scientific, Schwerte, Germany) on a Fluostar platereader (BMG labtech, Ortenberg, Germany) and quality checked by HS-Chips on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA). Up to 20 libraries were pooled and sequenced on an Illumina MiSeq platform with 2 x 250 bp read length using the Illumina MiSeq v2 reagents. After sequencing, basecalling and demultiplexing and FASTQ file generation was performed using a casava-based in house script.

DNA-Seq libraries were prepared from fragmented DNA (COVARIS S2, Woburn, Massachusetts, USA) according to recommendations made by the supplier (TruSeq DNA sample preparation v2 guide, Illumina, San Diego, CA, USA). Libraries were quantified by fluorometry, immobilised and processed onto a flow cell with a cBot followed by sequencing by synthesis by applying TruSeq v3 chemistry on a HiSeq2500 (all components by Illumina).
The de novo assembly of the reads was performed in CLC Genomics Workbench 7.0 (http://www.clcbio.com). The assembly data was exported as a BAM file, indexed using SAMtools [47 (link)] and imported in Gap5 [48 (link)]. Gaps were closed by PCR and primer-walking by applying dye-terminator sequencing performed on an ABI 310 capillary sequencer (Life technologies, Carlsbad, CA, USA).

Library preparation for all DNA pools was performed according to the Illumina TruSeq DNA Sample Preparation v2 Guide. DNA from each pool was fragmented by nebulization. After end repair and A-tailing, individual indexed paired end (PE) adapters were ligated to the DNA fragments which allowed multiplexed PE sequencing. The adapter-ligated fragments were size selected on a 2 % low melt agarose gel to a size of 500–800 bp. After enrichment PCR of fragments that carry adapters on both ends the final libraries were quantified by PicoGreen. The average fragment size of each library was determined on a BioAnalyzer High Sensitivity DNA chip. Samples from each of the six libraries were pooled in equimolar amounts and sequenced on a HiSeq1500 in rapid mode as well as in high output mode. Clusters on the flowcell for rapid runs were generated by on-board cluster generation using the TruSeq Rapid PE Cluster kit and sequenced according to the 2 × 151 PE scheme using TruSeq Rapid SBS chemistry. Cluster generation for high output runs was carried out on a cBot using the TruSeq PE Cluster kit v3, and sequenced according to the 2 × 101 scheme using the TruSeq SBS Kit v3. After completion of the sequencing runs, basecalling, demultiplexing and fastq file generation was performed using a CASAVA-based inhouse script.

For Seq-BSA, two DNA pools were developed by selecting the extreme cold-tolerant and extreme cold-sensitive individuals according to the PSST of the RIL population in the range from 0.50–0.95. The tolerant pool (T-pool) was made by mixing equal amounts of DNA from 20 extreme cold-tolerant RILs with a PSST above 0.90, and the sensitive pool (S-pool) was made by mixing equal amounts of DNA from 20 extreme cold-sensitive RILs with a PSST below 0.63 (Additional file 2: Table S1). The DNA isolated from DN422 and KY131 and the two DNA pools were prepared for sequencing.
Libraries for all the DNA pools were prepared according to the Illumina TruSeq DNA sample Preparation v2 Guide. The DNA libraries were sequenced on Illumina MiSeq platform using MiSeq Reagent Kit v2 (500 cycles) (Illumina Inc., San Diego, CA, USA). The short reads from both parents and the two DNA pools were aligned to Nipponbare reference genome (IRGSP 2005 (link)) using the BWA software (Li and Durbin 2009 (link)). Reads of the T-pool and S-pool were separately aligned to KY131 and DN422 consensus sequence reads to call SNPs with the SAM tools software (Li and Durbin 2009 (link)).

Library preparation for the DNA pools R1/B2444 and S1/B2446 was performed according to the Illumina TruSeq DNA Sample Preparation v2 Guide. DNA from each pool was fragmented by ultrasound shearing. After end repair and A-tailing, individual indexed PE adaptors were ligated to the DNA fragments, which allow a multiplexed PE sequencing run. The adaptor-ligated fragments were size selected on a 2% low melt agarose gel to a size of 350–650 bp. After enrichment, PCR of fragments of the final libraries that carry adaptors on both ends were quantified with a Qubit. The average fragment size of each library was determined on a BioAnalyzer High Sensitivity DNA chip. The samples were sequenced on a HiSeq-2000. Cluster generation for a high output run was done on a cBot using the TruSeq PE Cluster Kit v3, and 2 × 101 bp reads were generated using the TruSeq SBS Kit v3. Sequence read data of both pools were submitted to SRA. After completion of the sequencing runs, basecalling, demultiplexing and fastq file generation was performed using the CASAVA-1.8.2 programs. The results are summarized in Supplementary Table 2.