Improved Protocols for Illumina Sequencing

Bronner, Iraad F.; Quail, Michael A.; Turner, Daniel J.; Swerdlow, Harold

doi:10.1002/0471142905.hg1802s80

Cited by 100 publications

(94 citation statements)

References 20 publications

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“…This was followed by paired-end sequencing (2 × 100 bp) on a HiSeq 1500 platform, producing an average depth of 51× for diploid chromosomes; the European Nucleotide Archive accession number of these Nextera reads is ERP022358. (ii) TruSeq DNA library preparation was performed by shearing genomic DNA into 400- to 600-base-pair fragments (Covaris Adaptive Focused Acoustics technology), followed by generating 125-bp paired-end reads on the HiSeq 2000 version 4 according to the manufacturer’s standard sequencing protocol (39). Raw sequence data were deposited in the European Nucleotide Archive with accession number ERP017317; (iii) for all the Ld1S samples, the libraries were prepared using the KAPA hyper prep kit (Kapa Biosystems) with an input of 0.3 to 0.6 µg of sheared genomic DNA (Covaris E201) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression

Dumetz

Imamura

Sanders

et al. 2017

mBio

174

271

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Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.

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Section: Methodsmentioning

confidence: 99%

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression

Dumetz

Imamura

Sanders

et al. 2017

mBio

174

271

View full text Add to dashboard Cite

show abstract

“…A PCR product was deemed positive where there was an amplification band on the gel that was of the expected size (200-300 bp). PCR products were stored at -20 °C until they were pooled according to PCR plate to create sublibraries for purification with Mag-BIND ® RxnPure Plus magnetic beads (Omega Bio-tek Inc, GA, USA), following the double size selection protocol established by Bronner et al (2009). Ratios of 0.9x and 0.15x magnetic beads to 100 μL of each sub-library were used.…”

Section: Edna Metabarcoding Workflowmentioning

confidence: 99%

Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals

Harper

Handley

Carpenter

et al. 2019

Biological Conservation

115

112

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“…A NanoDrop 2000 (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) was used to assess DNA quality and concentration. Total DNA samples (400 ng) were used to construct sequencing libraries following the improved protocols for Illumina sequencing (10). In brief, genomic DNA samples were fragmented and ligated with Illumina standard adapters to both fragments ends.…”

Section: Methodsmentioning

confidence: 99%

Targeted molecular proﬁling of genetic alterations in colorectal cancer using next‑generation sequencing

et al. 2019

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Colorectal cancer (CRC) is a major contributor to cancer-associated mortality in China and remains a vast challenge worldwide. Although the genetic basis of CRC has been investigated, the uncommonly mutated genes in CRC remain unknown, in particular in the Asian population. In the present study, targeted region sequencing on 22 CRC and 10 paired non-cancerous tissues was performed to determine the genetic pattern of CRC samples in the Chinese population. Driver genes were detected by three distinct softwares, including MutSigCV, oncodriveFM and iCAGES. A total of 1,335 reliable somatic mutations were identified in tumour samples compared with normal samples. Furthermore, mismatch repair (MMR) mutant patients presented significantly higher mutation density compared with MMR wild-type patients. The results from MutSigCV, oncodriveFM and iCAGES analyses simultaneously detected 29 unique driver genes. In addition, the genes APC regulator of WNT signaling pathway, SMAD family member 4, neurofibromin 1, AT-rich interaction domain 5B and nuclear receptor corepressor 1 were the top five most frequently mutated genes in CRC samples, with mutation rates of 68, 36, 36, 32 and 27%, respectively. The findings from the present study may therefore serve as a basis for future investigation on the diagnosis and oncogenesis of CRC.

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Improved Protocols for Illumina Sequencing

Abstract: In this unit, we describe a set of improvements that have been made to the standard Illumina protocols to make the sequencing process more reliable in a high-throughput environment, reduce amplification bias, narrow the distribution of insert sizes, and reliably obtain high yields of data.

Cited by 100 publications

References 20 publications

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression

Modulation of Aneuploidy in Leishmania donovani during Adaptation to Different In Vitro and In Vivo Environments and Its Impact on Gene Expression

Environmental DNA (eDNA) metabarcoding of pond water as a tool to survey conservation and management priority mammals

Targeted molecular proﬁling of genetic alterations in colorectal cancer using next‑generation sequencing

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