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An internal fragment of 216 nucleotides within the PCR products generated for sequencing (positions 1223 1438 in the NS5B region according to the reference sequence H77, GenBank accession number NC004102) was used for phylogenetic analysis.
With the exception of the two small fragments generated for sequencing the M and NP segments, none of the sequencing amplicons is detectable in the respective qPCR assays (data not shown).
In general, the small RNA libraries generated for sequencing have complex RNA compositions.
Templated beads were generated for sequencing using standard manufacturers' protocols.
When the PCR products were generated for sequencing in the present study, a proof-reading polymerase was used to minimise procedure generated mutations.
Twenty-eight cDNA libraries were generated for sequencing and expression profile analysis: 20 were constructed from dehydrated plants, and 8 were generated from plants undergoing rehydration after a 2-h dehydration treatment (Table 1).
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For a given Pfam family, PSI-BLAST derived PSSM profiles were generated for sequences in 'Seed sequence dataset' using PSI-BLAST search against the sequences in seed alignment.
Biotinylated solution capture probe pools targeting the regions of interest were generated for sequence capture (Roche NimbleGen, Madison, WI) (Albert et al. 2007).
The annotation procedure (AnnotateBCL2 program; Figure 1B) starts from the entry templates generated for sequences that belong to the group of BCL-2 homologs or BH3-only proteins.
After amplification, the products were modified by random priming and extension to create double-stranded products suitable for generating libraries for sequencing.
T4 Rnl2 is now commonly used in RNA research, notably for the identification of small RNAs by generating libraries for sequencing with high-throughput sequencing technology.
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