Subsequently, DNA of the corresponding pools was converted into standard fragment libraries and sequenced in multiplex setup using barcodes that were introduced during library preparation.
Consideration of splice junction fragments removed biases created by enrichment for small RNAs or riboproteins that were introduced during library preparation for either SOLiD or Illumina platforms.
First, there were several steps where errors could have been introduced during library construction and amplification, such as the use of reverse transcriptase during first strand synthesis, or the 22-cycle exponential amplification of the reverse-transcribed products carried out prior to cloning (see Methods).
The increased level of duplicate reads seen at lower cell numbers is also assumed to be introduced during the PCR amplification (18 cycles) required as part of Illumina library preparation.
Essential to the analysis of miRNA sequencing data is removing any adapter sequences that were introduced during the library preparation as these impair proper genomic alignment.
We are not able to establish the exact source of these errors, but hypothesize that a portion of the errors are introduced during PCR library preparation.
To map the directional RNA-seq reads of H. pylori[ 24], we trimmed the polyA tails of the original datasets, which were introduced during the library preparation, and mapped the reads to the reference genome using Bowtie with the same parameter settings as for E. coli K12.
We suspected that GC bias was introduced during PCR in library preparation and cluster generation in the Illumina/Solexa sequencing workflow.
To minimize the effect of read pairs overlapping biotin junctions (the circularization junction point that is introduced during mate pair library construction) we only used those pairs with full-length genome alignment.
The RRE RNA samples used in this assay are barcoded within the SHAPE handle as described (Lucks et al., 2011) and additional barcodes were introduced during the PCR amplification step of library preparation using the NEBNext Multiplex Oligos for Illumina kit (NEB, Ipswich, MA, USA) to increase the multiplex capacity.
