Genome analyzer iix instrument

Sufficient diagnostic DNA was available from 11 CRLF2‐r patients (representitive of cohort and all patients have SNP analysis completed), which was prepared for library construction (300–500 bp), flow cell preparation and cluster formation using the Illumina no‐PCR library protocol (Illumina Inc, San Diego, CA), and 50 bp reads were performed using the Illumina Genome Analyzer IIx instrument following the manufactures guidelines. Structural variants were selected as previously reported.23Sufficient diagnostic and germ‐line DNA from the same 11 patients was prepared for Illumina paired‐end sequencing with subsequent exome enrichment using the Agilent SureSelect Human All Exon 50 MB kit (Agilent Technologies LTD, Berkshire, UK). Guidelines for the Illumina Genome Analyzer IIx instrument were followed for flow‐cell preparation, cluster generation and paired‐end sequencing of 75 bp reads.23, 24 Whole genome and exome sequencing data are available using EGA accession numbers EGAD00001002007 and EGAD00001002008, respectively.

Samples for deep sequencing analysis were prepared from 10 µg of each sample following Illumina's Small RNA sample preparation protocol (v1.5). During this process, samples were ligated with 3′ and 5′ adaptors, reverse transcribed and then amplified using PCR. Illumina Genome Analyzer IIx instrument (USA) was used for sequencing.
The data sequences were screened for the sequence of the small-RNA adaptor, and the adaptor sequences were trimmed using standard settings in Illumina's GAPipeline1.0. Processed Illumina data was managed by RandA software (Isakov et al., 2012 (link)). The reads were aligned to the human subset miRNAs in the miRbase database using BWA-aligner software. The number of reads was standardized by mapping each transcript according to its length and the initial total number of mapped reads in the sample based on the ‘reads per kilo-base per million’ (RPKM) method (Mortazavi et al., 2008 (link)). Only perfect matches were counted in the main analysis. Next, results were ranked in terms of differentially expressed miRNAs between the two samples. Statistical analysis was performed using a chi-square distribution.

DNA samples (180 ng) of CS and M808 were digested first with PstI (New England BioLabs, Inc., Ipswich, MA, USA) at 37°C for 1 h and then with BstNI (New England BioLabs) at 60°C for 1 h. We selected this combination of restriction enzymes based on in silico analysis in rice, our experience in sugarcane,^{17 ,18} and its use in barley.^{4 (link)} Digested DNA was ligated to PstI adapters (5′-CACGATGGATCCAGTGCA-3′ and 5′-CTGGATCCATCGTGCA-3′) using T4 DNA ligase (Nippon Gene, Tokyo, Japan) and ATP (Wako Pure Chemical Industries, Osaka, Japan). The ligation reaction was run at 16°C for 16 h and the ligase was inactivated by holding at 60°C for 20 min.
Ligated samples were PCR-amplified for 30 cycles using 5′-GATGGATCCAGTGCAG-3′ with PrimeSTAR HS DNA Polymerase (Takara Bio, Shiga, Japan) under the following conditions: 10 s at 98°C, 15 s at 55°C, and 60 s at 72°C. PCR products were purified using a MinElute PCR Purification Kit (Qiagen, Hilden, Germany). The constructed libraries were sequenced as 2 × 100 nt paired-end reads on the Genome Analyzer IIx instrument (Illumina, San Diego, CA, USA). The sequences were deposited to the DNA Data Bank of Japan (DDBJ) Short Read Archive (DRA001257).