Gaiix instrument

The genomic DNA was purified using phenol-chloroform extraction and ethanol precipitation from each culture at appropriate time points. In the case of the feasibility study regarding the DNA copy number gradient during different growth phases (Fig. 2), the sequence library was prepared using the standard protocol for the KAPA HyperPlus Kit (for Illumina), and sequencing was performed with a NextSeq 500 instrument (Illumina, Inc.) using a 75 bp single-end read. For the eRP demonstration (Fig. 3), the sequence library was prepared using a standard protocol with the Nextera DNA Library Preparation Kit (Illumina, Inc.), and sequencing was performed with a GAIIx instrument (Illumina, Inc.) using a 100 bp paired-end read. All reads were used for each assembly. The quality of the sequencing results was assessed with FastQC (v0.10.1) [40 ]. The data sets obtained from this study were deposited and are available at the DNA Data Bank of Japan (DDBJ: http://www.ddbj.nig.ac.jp/) Sequence Read Archive with Accession no. DRA005896 (Additional file 8: Table S6).

15 µl serums from each of 6 patients including 3 treated with ERT for more than one year and 3 without ERT were incubated with HTG lysis buffer and Proteinase K (Ambion) at 20 °C for 2 hours. The sample plates were then loaded into an HTG Edgeseq Processor. After the automated preparation process, library were prepared with TruSeq Small RNA Prep kit (Illumina) according to the manufacturer’s instruction. Single-end reads of 51 bp in length were then sequenced on an Illumina GAIIx instrument. For expression level quantification, trimmed reads were mapped to the genome reference (hg19) allowing one mismatch and quantified applying Avadis NGS software (v1.4). Reads mapped to multiple locations in the genome were removed from further quantification. Annotation from miRBase v20 were used to designate reference mapped reads to miRNAs.

To construct different insert-size libraries, genomic DNA was separated by agarose gel electrophoresis, and regions containing DNA of about 300 bp, 350 bp and 400 bp length were cut out by reference to their corresponding size markers. Libraries for genomic DNA sequencing were prepared using a Paired-End DNA Sample Prep Kit (Illumina) according to the manufacturer's instructions. The resultant libraries were sequenced (2 x 150 cycles paired-end) on an Illumina GAIIx instrument.

A barcoded library was prepared from each sperm or subcell aliquot of the Angus animals using the ABI SOLiD4 fragment library kit following the manufacturer’s instructions (Life Technologies, Foster City, USA). Fragment reads of 75 bp were obtained using ABI 5500 instruments and the exact call chemistry (ECC) module. ECC makes it possible to convert the color space data to nucleotide base sequences prior to mapping.
A separate library was prepared from DNA extracted from 50 cells from the Angus steer, which was amplified with Genomiphi and sequenced on the ABI SOLiD 5500 (Life Technologies, Foster City, USA). A library was also prepared from whole genomic DNA of the Angus steer using the Illumina PE-102–1001 paired end library kit and 2×100 bp reads were obtained with an Illumina GAIIx instrument (Illumina, San Diego, USA).
Libraries from the EAZ samples were prepared using the Illumina TrueSeq protocol and 2 × 100 bp reads were obtained on an Illumina HiSeq2000.
The fosmid library was aliquoted into 192 pools each predicted to contain ~6000 unique clones. Three pools of 6000 clones were merged to make each of 47 libraries using the Illumina TruSeq Paired End library preparation kit and 2 × 100 bp paired end sequence reads were obtained on an Illumina HiSeq2000.

DNA was extracted from 1 culture each of strain N7153 and N7182 grown in LB broth to stationary phase using Qiagen 100/G genomic tips from 5 ml cultures following the manufacturer's protocol. Genome sequencing was performed using an Illumina GAIIx instrument with 100 bp paired end reads. Paired reads were trimmed to remove adapters and mapped against the E. coli K12 strain MG1655 NC_000913 genome using bwa (57 (link)), duplicates marked using Picard (http://broadinstitute.github.io/picard) and variant analysis performed with SAMtools (58 (link)), followed by merging of variant tables using perl. Identified high quality synonymous and non-synonymous single nucleotide polymorphisms were annotated manually using the Integrative Genomics Viewer (59 (link)). The Illumina data were submitted in the form of fastq files to the European Nucleotide Archive (ENA) and are available under accession number PRJEB14483 at http://www.ebi.ac.uk/ena/data/view/PRJEB14483.

Blood samples from patients were plated and single colonies picked and stored at −80 °C until processing. Samples were grown on trypticase soy agar (TSA) (BD, Franklin Lakes, USA) at 37 °C for 24 h, after which DNA was extracted using the Qiagen DNeasy Blood and Tissue Purification kit including a pre-lysis step using lysostaphin for Gram-positive extractions according to the manufacturer’s recommendations (Qiagen, Valencia, CA, USA). DNA was prepared for multiplexed, paired-end sequencing (2×100 bp) on an Illumina GA_IIX instrument using the Library Preparation kit with standard PCR library amplification (KAPA Biosystems, Woburn, MA, USA) as described previously [22 (link)]. Additionally, six samples that failed initial sequencing were resequenced on an Illumina MiSeq instrument using 2×250 V2 technology (Table S1). The average coverage across all 100 isolates was 130×. Genomes were assembled with UGAP (https://github.com/jasonsahl/UGAP), which utilizes SPAdes v3.10.1 [23 (link)] and Pilon [24 (link)] post-assembly error correction. MLST was performed with the raw reads using SRST2 [25 (link)].

We determined the circulating miRNA profile in the procured human plasma specimens by performing the HTG EdgeSeq miRNA Whole Transcriptome Assay (miRNA WTA) (HTG Molecular Diagnostics, Inc. Tucson, AZ). The HTG EdgeSeq miRNA WTA enables us to measure the expression of 2,083 human miRNA transcripts using next-generation sequencing (NGS). The plasma samples (20 µl) were incubated with 20 µl plasma lysis buffer plus 4 µl of proteinase-K (miRNA lysis buffer kit; HTG Molecular Diagnostics) with constant shaking at 50֯C for 3 h to denature them. The miRNA expression profile was determined as per standardized protocols [6 (link)]. Briefly, the samples were loaded into an HTG Edgeseq Processor. After the automated preparation process, libraries were prepared with TruSeq Small RNA Prep kit (Illumina). Single-end reads of 51 bp in length were sequenced using an Illumina GAIIx instrument. To quantify the expression levels, trimmed reads were mapped to the genome and duplications were removed. Finally, the annotation from miRBase v20 was used to designate reference mapped reads to mature miRNAs.