title: Strategies for achieving high sequencing accuracy for low diversity samples and avoiding sample bleeding using illumina platform.
abstract: Sequencing microRNA, reduced representation sequencing, Hi-C technology and any method requiring the use of in-house barcodes result in sequencing libraries with low initial sequence diversity. Sequencing such data on the Illumina platform typically produces low quality data due to the limitations of the Illumina cluster calling algorithm. Moreover, even in the case of diverse samples, these limitations are causing substantial inaccuracies in multiplexed sample assignment (sample bleeding). Such inaccuracies are unacceptable in clinical applications, and in some other fields (e.g. detection of rare variants). Here, we discuss how both problems with quality of low-diversity samples and sample bleeding are caused by incorrect detection of clusters on the flowcell during initial sequencing cycles. We propose simple software modifications (Long Template Protocol) that overcome this problem. We present experimental results showing that our Long Template Protocol remarkably increases data quality for low diversity samples, as compared with the standard analysis protocol; it also substantially reduces sample bleeding for all samples. For comprehensiveness, we also discuss and compare experimental results from alternative approaches to sequencing low diversity samples. First, we discuss how the low diversity problem, if caused by barcodes, can be avoided altogether at the barcode design stage. Second and third, we present modified guidelines, which are more stringent than the manufacturer's, for mixing low diversity samples with diverse samples and lowering cluster density, which in our experience consistently produces high quality data from low diversity samples. Fourth and fifth, we present rescue strategies that can be applied when sequencing results in low quality data and when there is no more biological material available. In such cases, we propose that the flowcell be re-hybridized and sequenced again using our Long Template Protocol. Alternatively, we discuss how analysis can be repeated from saved sequencing images using the Long Template Protocol to increase accuracy.
authors: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, 301 University Blvd, Galveston, TX, 77555, USA; Institute for Translational Sciences, University of Texas Medical Branch at Galveston, 301 University Blvd, Galveston, TX, 77555, USA.
authors: Abhishek
authors: A
authors: Mitra
authors: Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland.
authors: Magdalena
authors: M
authors: Skrzypczak
authors: Laboratory of Bioinformatics and Systems Biology, Centre of New Technologies, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland.
authors: Krzysztof
authors: K
authors: Ginalski
authors: Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, 301 University Blvd, Galveston, TX, 77555, USA; Institute for Translational Sciences, University of Texas Medical Branch at Galveston, 301 University Blvd, Galveston, TX, 77555, USA; Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, 301 University Blvd, Galveston, TX, 77555, USA.
authors: Maga
authors: M
authors: Rowicka
mesh_terms: D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D035683:MicroRNAs / Q000032:analysis*; D010363:Pattern Recognition, Automated; D012107:Research Design; D017423:Sequence Analysis, RNA; D012984:Software
chemical_list: D035683:MicroRNAs