Affordable, accurate and unbiased RNA sequencing by manual library miniaturization: A case study in barley.
RNA sequencing
library preparation
methods and techniques
miniaturization
plant breeding
plant genetics
Journal
Plant biotechnology journal
ISSN: 1467-7652
Titre abrégé: Plant Biotechnol J
Pays: England
ID NLM: 101201889
Informations de publication
Date de publication:
11 2023
11 2023
Historique:
revised:
12
05
2023
received:
16
11
2022
accepted:
01
07
2023
medline:
23
10
2023
pubmed:
18
8
2023
entrez:
18
8
2023
Statut:
ppublish
Résumé
We present an easy-to-reproduce manual miniaturized full-length RNA sequencing (RNAseq) library preparation workflow that does not require the upfront investment in expensive lab equipment or long setup times. With minimal adjustments to an established commercial protocol, we were able to manually miniaturize the RNAseq library preparation by a factor of up to 1:8. This led to cost savings for miniaturized library preparation of up to 86.1% compared to the gold standard. The resulting data were the basis of a rigorous quality control analysis that inspected: sequencing quality metrics, gene body coverage, raw read duplications, alignment statistics, read pair duplications, detected transcripts and sequence variants. We also included a deep dive data analysis identifying rRNA contamination and suggested ways to circumvent these. In the end, we could not find any indication of biases or inaccuracies caused by the RNAseq library miniaturization. The variance in detected transcripts was minimal and not influenced by the miniaturization level. Our results suggest that the workflow is highly reproducible and the sequence data suitable for downstream analyses such as differential gene expression analysis or variant calling.
Identifiants
pubmed: 37593840
doi: 10.1111/pbi.14126
pmc: PMC10579711
doi:
Substances chimiques
RNA
63231-63-0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
2241-2253Informations de copyright
© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
Références
Bioinformatics. 2015 Jan 15;31(2):166-9
pubmed: 25260700
BMC Genomics. 2018 Jul 13;19(1):531
pubmed: 30001700
J Biomol Tech. 2020 Dec;31(4):151-156
pubmed: 33100919
Plant Methods. 2011 Mar 13;7:7
pubmed: 21396125
Nat Rev Genet. 2019 Nov;20(11):631-656
pubmed: 31341269
Front Plant Sci. 2017 May 01;8:647
pubmed: 28507554
PLoS One. 2019 Jan 10;14(1):e0206194
pubmed: 30629604
Clin Infect Dis. 2021 Feb 1;72(3):421-422
pubmed: 32255494
Genome Res. 2019 Nov;29(11):1816-1825
pubmed: 31519740
Nat Methods. 2011 Nov 20;9(1):72-4
pubmed: 22101854
Nat Protoc. 2012 Apr 05;7(5):813-28
pubmed: 22481528
Front Plant Sci. 2012 Aug 28;3:202
pubmed: 22973283
Cell. 2015 May 21;161(5):1202-1214
pubmed: 26000488
STAR Protoc. 2020 Sep 18;1(2):100081
pubmed: 33000004
Nat Methods. 2015 Apr;12(4):323-5
pubmed: 25730492
BMC Bioinformatics. 2017 Mar 14;18(Suppl 3):43
pubmed: 28361665
Bioinformatics. 2014 Aug 1;30(15):2114-20
pubmed: 24695404
BMC Genomics. 2015 Aug 05;16:578
pubmed: 26238335
Nat Methods. 2013 Nov;10(11):1096-8
pubmed: 24056875
J Genet. 2013 Apr;92(1):155-61
pubmed: 23640422
Nat Biotechnol. 2019 Aug;37(8):907-915
pubmed: 31375807
Biotechniques. 2012 Feb;52(2):87-94
pubmed: 22313406
J Mol Diagn. 2020 May;22(5):720-727
pubmed: 32142899
G3 (Bethesda). 2020 Jan 7;10(1):143-150
pubmed: 31676507
Am J Hum Genet. 2021 Oct 7;108(10):1880-1890
pubmed: 34478634
Front Plant Sci. 2021 Jul 15;12:629089
pubmed: 34335638
Wiley Interdiscip Rev Dev Biol. 2021 Sep;10(5):e396
pubmed: 32940008
Genetics. 2009 Dec;183(4):1525-34
pubmed: 19805816
Nat Biotechnol. 2020 Jun;38(6):747-755
pubmed: 32518403
Sci Rep. 2015 Apr 01;5:9570
pubmed: 25831155
Sci Rep. 2016 May 09;6:25533
pubmed: 27156886
Nat Commun. 2018 Jul 26;9(1):2937
pubmed: 30050112
Bioinformatics. 2016 Oct 1;32(19):3047-8
pubmed: 27312411
BMC Genomics. 2020 Oct 20;21(1):729
pubmed: 33081689
J Pers Med. 2016 Mar 25;6(2):
pubmed: 27023617
Bioinformatics. 2010 Jan 1;26(1):139-40
pubmed: 19910308
BMC Genomics. 2014 Oct 20;15:912
pubmed: 25331572
BMC Genomics. 2019 Oct 29;20(1):787
pubmed: 31664921
Front Genet. 2021 Jun 28;12:655707
pubmed: 34262593
Genes (Basel). 2020 Nov 23;11(11):
pubmed: 33238469
BMC Bioinformatics. 2016 Oct 8;17(1):419
pubmed: 27717304
Genome Biol. 2019 Apr 19;20(1):71
pubmed: 30999927
Proc Natl Acad Sci U S A. 2005 Apr 5;102(14):5084-9
pubmed: 15784739
Nature. 2015 Oct 1;526(7571):68-74
pubmed: 26432245
J Lab Autom. 2016 Aug;21(4):557-67
pubmed: 26891732
Clin Chem. 2019 Feb;65(2):272-281
pubmed: 30523199
Sci Rep. 2022 Mar 8;12(1):4091
pubmed: 35260714
BMC Biol. 2014 May 30;12:42
pubmed: 24885439
Genome Res. 2019 Nov;29(11):1826-1835
pubmed: 31649055
BMC Genomics. 2016 Jun 13;17:458
pubmed: 27297323
Nature. 2011 Feb 10;470(7333):198-203
pubmed: 21307932
Bioinformatics. 2009 Aug 15;25(16):2078-9
pubmed: 19505943
Genome Biol. 2016 Apr 28;17:77
pubmed: 27121950
Plant Biotechnol J. 2022 Apr;20(4):676-690
pubmed: 34783155
BMC Bioinformatics. 2021 Aug 25;22(Suppl 9):403
pubmed: 34433407
Comput Struct Biotechnol J. 2020 Jun 12;18:1569-1576
pubmed: 32637053
Am J Hum Genet. 2013 Oct 3;93(4):641-51
pubmed: 24075185
Cold Spring Harb Perspect Med. 2019 Nov 1;9(11):
pubmed: 30478097