Using high-throughput bisulfite sequencing on the Genome Analyzer, the authors show widespread DNA demethylation in Arabidopsis thaliana endosperm. This was found to be at least partially controlled by the maternal DNA glycosylase DEMETER as mutations of this somewhat restored CG methylation in this area.

Genes that code for proteins involved in innate immunity show gains and losses across insect taxa. The authors used short read sequencing of olido(dT)-primed double-stranded cDNA, carried out on the Genome Analyzer II system, to compare infection response of Drosophila melanogaster and the distantly related Drodophila virilis at the transcription level. There were a number of differences between the two, highlighting evolutionarily novel transcription data in D. virilis. They also showed differentially expressed genes between infected and non-infected D. virilis.

The authors analyzed and compared microRNAs (miRNAs) in the temperate grass Brachypodium distachyon. Using deep sequencing technology on the Genome Analyzer, the authors identified 23 novel miRNAs in Chinese Spring wheat and 94 conserved and one novel miRNA in Brachypodium. They also investigated miRNA expression levels and found differences between Brachypodium tissue types (vegetative and reproductive) and between the two plant types for some mRNAs, particularly one involved in control of lignin metabolism.

The authors used high-throughput sequencing with the Genome Analyzer to genotype recombinant populations. Using 150 rice recombinant inbred lines (RILs) they collectively assessed single nucleotide polymorphisms (SNPs) using a sliding window approach that encompasses 15 SNPs at once. Genotype calling involved examination of SNP number ratios from the parents. They could determine recombination breakpoints between two SNPs and constructed very accurate recombination maps of the RILs. This method had large time, cost, accuracy, and resolution advantages over PCR.

Paired-end sequencing using the Genome Analyzer, and SNP genotyping using the Human1M-duo DNA analysis BeadChip, helped investigate a Korean male’s genome. Over 420,000 novel SNPs and 21 homozygous alleles were found. More SNPs were shared with a Chinese male (60%), then two Caucasian and one African male (50%, 53% and 56% respectively). Of 343,000 indels, one-third were within genes. Indel similarity was almost 100% between the Asian males, less for the African and Caucasian ones. Deletion structural variants were in 21 coding genes. Nearly 6% of reads were novel.

The authors used high-throughput parallel sequencing, on the Genome Analyzer, of small interfering RNAs (siRNAs) to identify both known and unknown RNA and DNA viruses infecting sweet potato plants. These included RNA, ssDNA, and dsDNA reverse transcribing viruses. They also reconstruct the entire genome of sweet potato feathery mottle virus (SPFMV).

Genomic structural variations (SVs) include insertions, deletions, and inversions. The authors describe two algorithms—the VariationHunter-Set Cover method and the VariationHunter-Probabilistic method—to analyze SVs of a genome sequence obtained using next generation-based, paired-end, whole genome shotgun-sequencing, using the Genome Analyzer, compared to a reference genome. They find the algorithms to be fast and reliable and report they can also be used for capillary sequencing technology.

Epigenetic DNA methylation predominantly occurs at cytosine residues in the CpG dinucleotide. Sodium bisulfite-related analysis relies on it’s preserving of methylated cytosines Investigation of genome-wide DNA methylation in the mouse (Mus musculus) was carried out through optimization of reduced representation bisulfite sequencing (RRBS), which generates nucleotide resolution Illumina-based libraries using the Genome Analyzer. RRBS is similar to BS-seq but it used CpG-specific endonucleases to digest genomic DNA and provides enhanced coverage.

Piwi-interacting RNAs (piRNAs) are transposon silencers that act as guides for a subfamily of Argonaute proteins—PIWI proteins—expressed in germline cells. The authors used flies with ago3 loss-of-function mutations to show that Ago3 increases levels of these piRNAs. The also show a separate, simpler, somatic, piRNA pathway that does not rely on Ago3 amplification. They used the Genome Analyzer for small RNA sequencing.

Genes residing in the chromosomal region suspected to be linked with the neurological disorder sensory/motor neuropathy with ataxia (SMNA) were investigated. Using massively parallel sequencing on the Genome Analyzer, and other methods of analysis, they found a nonsynonymous variant in the interferon-related developmental regulator gene 1 (IFRD1) in the suspect region (7q22-q32), which was not found in healthy controls. This gene is known to be localized to brain and spinal cord areas commonly affected in SNMA.

Short sequence fragments generated by RNA-Seq can be used to measure gene expression levels and gene splice variants. TopHat is an open-source software package with a rapid processing time that uses large-scaled mapping of RNA-Seq reads, such as those generated by the Genome Analyzer system. It can identify novel transcripts and provide an estimate of their abundance.

To investigate their role in a more open chromatin structure, the authors examined locations of histone variants H2AZ, H2BV, H3V, and H4V, along with BDF3, using ChIP and ChIP-seq (Genome Analyzer) in Trypanosome brucei. They found these increased at probable transcription start sites (TSS) (some of which were previously unknown) of the RNA polymerase II (Pol II) gene, indicating a role in transcription. They also found appearance of H2AZ and H2BV at a nucleosome made it less stable.

The authors adapted a molecular inversion probe (MIP) protocol to enable “simultaneous amplification and accurate shotgun sequencing of 50,000 exons,” using the Genome Analyzer. Of these targets, 91% were captured, compared to 18% previously. They also examined a method to directly sequence MIP amplicons and found a greater proportion of mapped reads compared to shotgun sequencing (90.4% vs 56.8%), with almost 100% specificity. This eliminated the requirement for shotgun library construction, and only needed submicrogram amounts of DNA.

Without food after hatching Caenorhabditis elegans larvae enter L1 arrest. Utilizing the Genome Analyzer II, the authors found starved larvae had a quicker gene expression response during subsequent feeding than fed larvae during subsequent starvation. They found RNA polymerase II (Pol II) transcribed starvation-response genes during L1 arrest with the enzyme centered around growth/development genes. Decreased accumulation following feeding suggested Poll II was a priming factor for the rapid gene response following starvation.