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A benchmark of hemoglobin blocking during library preparation for mRNA-Sequencing of human blood samples.

ABSTRACT: RNA-Sequencing (RNA-Seq) of peripheral blood can be a valuable source of information for investigating the status and mechanism of diseases. However, blood contains 50-80% unwanted hemoglobin (Hb) transcripts. Lexogen's QuantSeq mRNA-Seq-Kit for Illumina RNA-Seq features a 'Globin Block' (GB) module that depletes Hb cDNAs during library preparation. Here, we aimed to assess GB's effectiveness and checked for technical biases attributable to GB. Using whole blood total RNA samples of 91 healthy individuals, we sequenced 91 pairs of GB and non-blocked samples (noGB) on Illumina HiSeq2500 and 8 pairs of GB/noGB technical replicates on HiSeq4000. GB reduced the fraction of Hb transcripts from 43% (s.d. 14%) to 8.0% (s.d. 4.3%). From GB samples we detected 1,397 more expressed genes at approximately 11 million reads per RNA-isolate. Enrichment and differential expression analyses did not reveal significant differences for GB and noGB samples with respect to molecular function. In contrast to results from studies that have examined the performance of GB during RNA isolation, we were able to assign GB to corresponding noGB samples (from multiple sequencing runs on HiSeq2500) with at least 89.8% accuracy from the complete correlation matrix of all GB/GB, noGB/noGB and GB/noGB pairs. However, the use of different sequencers (HiSeq2500 vs HiSeq4000) impaired assignment of technical replicates, whereas assignment of GB to corresponding noGB samples worked perfectly when sequencing on one lane on HiSeq4000. Lexogen's GB RNA-Seq module is a valuable addition during mRNA-Seq library preparation which works even with low amounts of input total RNA (50?ng per sample). GB facilitated the detection of low abundant transcripts and yielded more non-hemoglobin reads, while preserving biological information. We observed that differences in sequencing run and platform have a far greater effect on technical variation than the use of GB.

SUBMITTER: Uellendahl-Werth F

PROVIDER: S-EPMC7101437 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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A benchmark of hemoglobin blocking during library preparation for mRNA-Sequencing of human blood samples.

Uellendahl-Werth Florian F Wolfien Markus M Franke Andre A Wolkenhauer Olaf O Ellinghaus David D

Scientific reports 20200327 1

RNA-Sequencing (RNA-Seq) of peripheral blood can be a valuable source of information for investigating the status and mechanism of diseases. However, blood contains 50-80% unwanted hemoglobin (Hb) transcripts. Lexogen's QuantSeq mRNA-Seq-Kit for Illumina RNA-Seq features a 'Globin Block' (GB) module that depletes Hb cDNAs during library preparation. Here, we aimed to assess GB's effectiveness and checked for technical biases attributable to GB. Using whole blood total RNA samples of 91 healthy i ...[more]

PMID: 32221409

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Project description:BackgroundmRNA sequencing is a powerful technique, which is used to investigate the transcriptome status of a gene of interest, such as its transcription level and splicing variants. Presently, several RNA sequencing (RNA-Seq) methods have been developed; however, the relative advantage of each method has remained unknown. Here we used three commercially available RNA-Seq library preparation kits; the traditional method (TruSeq), in addition to full-length double-stranded cDNA methods (SMARTer and TeloPrime) to investigate the advantages and disadvantages of these three approaches in transcriptome analysis.ResultsWe observed that the number of expressed genes detected from the TeloPrime sequencing method was fewer than that obtained using the TruSeq and SMARTer. We also observed that the expression patterns between TruSeq and SMARTer correlated strongly. Alternatively, SMARTer and TeloPrime methods underestimated the expression of relatively long transcripts. Moreover, genes having low expression levels were undetected stochastically regardless of any three methods used. Furthermore, although TeloPrime detected a significantly higher proportion at the transcription start site (TSS), its coverage of the gene body was not uniform. SMARTer is proposed to be yielded for nonspecific genomic DNA amplification. In contrast, the detected splicing event number was highest in the TruSeq. The percent spliced in index (PSI) of the three methods was highly correlated.ConclusionsTruSeq detected transcripts and splicing events better than the other methods and measured expression levels of genes, in addition to splicing events accurately. However, although detected transcripts and splicing events in TeloPrime were fewer, the coverage at TSS was highest. Additionally, SMARTer was better than TeloPrime with regards to the detected number of transcripts and splicing events among the understudied full-length double-stranded cDNA methods. In conclusion, for short-read sequencing, TruSeq has relative advantages for use in transcriptome analysis.

Dataset Information

A benchmark of hemoglobin blocking during library preparation for mRNA-Sequencing of human blood samples.

Publications

A benchmark of hemoglobin blocking during library preparation for mRNA-Sequencing of human blood samples.

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