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trimLinDA: v0.4

Single-tube linear DNA amplification (LinDA) for genome-wide studies using a few thousand cells

Pattabhiraman Shankaranarayanan, Marco-Antonio Mendoza-Parra, Wouter van Gool, Luisa M. Trindade and Hinrich Gronemeyer

Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells (1,2). LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation combined with massive parallel sequencing (ChIP-seq) has been done for transcription factors and epigenetic modification of chromatin histones with 1,000 to 5,000 cells. LinDA largely simplified reChIP-seq experiments to monitor co-binding at chromatin target sites (3). The single tube design of LinDA is ideal for handling ultra-small amounts of DNA (<30pg) and is compatible with automation. The actual hands-on working time is less than 6h with one overnight reaction. A detailed LinDA protocol describing all materials and critical steps, in addition to examples and controls is available as part of the corresponding Nature Protocols publication (2).

TrimLinDA: a script for computational removal of polyA stretches

While LinDA-amplified libraries are sequenced following standard procedures, the alignment of sequenced reads to the reference genome may require particular attention. LinDA incorporates oligo(A) sequences in the 5'-ends of the amplified fragments, most of which are removed by BpmI cleavage. However, the tailing reaction cannot be precisely controlled and may generate oligo(A) sequences extending the 15 As of the T7-BpmI-oligo(A)15 primer. Therefore, a certain fraction of reads may contain oligo(A) stretches which cannot be properly aligned to the reference genome and reduce the number of mappable reads.

To circumvent this problem, we provide a perl script, trimLinDA, to computationally remove the residual polyA stretches from sequencing data generated with the LinDA protocol.

TrimLinDA provides two ways for removing the polyA stretches from a fastq file:

1. static trimming mode: a fixed number of nucleotides are removed from the 5'-end of the sequenced reads)

2. dynamic trimming mode: the number of removed nucleotides per sequenced read depends only on the number of identified 5' polyA-stretches. In addition reads with length <20nts after trimming are discarded to avoid multiple mapping

For more information how to use the trimLinDA script, please download and extract the zip archive and follow the instructions in the included README file.  

REFERENCES:

1 Single-tube linear DNA amplifi-
cation (LinDA) for robust ChIP-seq.

Nature Methods. 2011 Jun 5;8(7):565-7

2 Single-tube linear DNA amplifi-
cation (LinDA) for genome-wide studies using a few thousand cells.

Nature Protocols (manuscript in
revision)

3. Sequential chromatin immunoprecipitation protocol for global analysis through massive parallel sequencing (reChIP-seq)
Protocol exchange, DOI 10.1038/protex.2011.256