Whole-Exome Sequencing for Congenital Lymphoedema

Whole-exome sequencing was performed on DNA extracted from whole blood using a previously described method with the following alterations: the Agilent SureSelectXT Human All Exon V4 kit (Agilent Technologies) was used27 (link), and sequencing was performed on the HiSeq2500 instrument (Illumina) with 100-bp paired-end sequencing. Sequence was aligned to the human reference genome (Hg19) using Burrow–Wheeler Aligner (0.5.11)28 (link); variants were called with Samtools29 (link) and Genome Analysis Toolkit (v.1.6)30 (link) and annotated with Annovar31 (link). Variants with a quality score <10 were excluded from analysis. Variant filtering and segregation was performed with in-house developed software (https://github.com/brendanofallon/VarViewer). All genes previously associated with congenital lymphoedema (CCBE1, FOXC2, FLT4, KIF11, GATA2, GJC2, SOX18, FAT4 and PTPN14) were specifically analysed and no rare or pathogenic variants were found in any family member despite full sequencing coverage. The reported PIEZO1 variants were confirmed by Sanger sequencing using standard methods. Primer sequences are available upon request.

Free full text: Click here

Lukacs V., Mathur J., Mao R., Bayrak-Toydemir P., Procter M., Cahalan S.M., Kim H.J., Bandell M., Longo N., Day R.W., Stevenson D.A., Patapoutian A, & Krock B.L. (2015). Impaired PIEZO1 function in patients with a novel autosomal recessive congenital lymphatic dysplasia. Nature Communications, 6, 8329.

Publication 2015

Agilent SureSelectXT Human All Exon V4 kit HiSeq2500

Blood Congenital lymphoedema Exon Family member Flt4 Gata2 Genes Genome Genome human Human Pathogenic Primer Ptpn14 Variant

Corresponding Organization :

Other organizations : Scripps Research Institute, Howard Hughes Medical Institute, Genomics Institute of the Novartis Research Foundation, ARUP Laboratories (United States), University of Utah

Top 5 similar protocols

Variable analysis

independent variables

DNA extraction method
Exome capture kit (Agilent SureSelectXT Human All Exon V4)
Sequencing platform (HiSeq2500 instrument)
Sequencing read length (100-bp paired-end)

dependent variables

Variants detected and called
Variant quality score
Presence or absence of rare or pathogenic variants in specific genes (CCBE1, FOXC2, FLT4, KIF11, GATA2, GJC2, SOX18, FAT4, PTPN14, PIEZO1)

control variables

Reference genome (Hg19)
Alignment tool (Burrows-Wheeler Aligner)
Variant calling tools (Samtools, Genome Analysis Toolkit)
Variant annotation tool (Annovar)
Variant filtering and segregation tool (in-house developed software)

positive controls

Sanger sequencing to confirm the reported PIEZO1 variants

negative controls

None explicitly mentioned

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!