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RNA-seq03:21

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RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while...
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Detection of Rare Genomic Variants from Pooled Sequencing Using SPLINTER
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Identification and quantification of chimeric sequencing reads in a highly multiplexed RAD-seq protocol.

Maria Luisa Martin Cerezo1,2, Rohan Raval1, Bernardo de Haro Reyes1,2

  • 1Department of Biological and Geographical Sciences, School of Applied Sciences, University of Huddersfield, Huddersfield, UK.

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|June 7, 2022
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Summary

This study investigated index hopping and PCR chimeras in highly multiplexed genomic sequencing. Type B libraries (pooled PCRs) showed more misassigned reads than Type A (individual PCRs), impacting data accuracy.

Keywords:
RAD-seqadaptersbarcodeschimerasindex hoppingquaddRADread misassignment

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Highly multiplexed sequencing improves cost-effectiveness in genomic studies.
  • Combinatorially barcoded adapters enable genotyping of many individuals.
  • Potential for misassigned reads to incorrect samples exists in multiplexed strategies.

Purpose of the Study:

  • To analyze index hopping and PCR chimeras in highly multiplexed sequencing.
  • To compare misassigned read rates between two library preparation methods.
  • To quantify chimeric sequences in RAD-seq protocols.

Main Methods:

  • Modified quaddRAD protocol used for analysis.
  • Two library types created: Type A (individual PCRs) and Type B (pooled PCRs).
  • Fixed pairs of inner barcodes identified chimeric reads.

Main Results:

  • Type B libraries exhibited a higher percentage of misassigned reads (1.15%) than Type A libraries (0.65%).
  • Chimeric sequences constituted up to 1.56% (Type A) and 1.29% (Type B) of reads.
  • Allowing >2 mismatches for barcode rescue significantly increased chimeric read recovery.

Conclusions:

  • Library preparation method impacts misassigned read rates in multiplexed sequencing.
  • Chimeric sequences are a significant, often undetected, source of error.
  • Recommendations provided for optimizing multiplexed RAD-seq protocols and data analysis to minimize and quantify chimeric sequences.