Blood samples were collected from 87 of 122 patients. Genomic DNA was extracted from the peripheral blood leukocytes using Qiagen DNA Blood kit (Qiagen, Hilden, Germany). A gene panel (NimblegenSeqCap EZ system, Roche, Basel, Switzerland) was designed to capture all exons and 10 bp flanking intron sequences of the 31 CHH-related genes (Supplementary Table 1
Nimblegenseqcap ez system
Manufactured by Roche
Sourced in Switzerland, United States
The NimblegenSeqCap EZ system is a targeted DNA enrichment solution used for next-generation sequencing (NGS) applications. It enables the selective capture and sequencing of specific genomic regions of interest. The system includes reagents and protocols for the efficient enrichment of targeted regions, facilitating focused and cost-effective sequencing.
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Lab products found in correlation
Agencourt rnadvance blood kit, by Beckman Coulter (3 mentions)
Neb second strand synthesis kit, by New England Biolabs (3 mentions)
Superscript 3, by Thermo Fisher Scientific (3 mentions)
Dna blood kit, by Qiagen (2 mentions)
Hiseq 2500 sequencing system, by Illumina (2 mentions)
Kapa hifi hotstart, by Roche (2 mentions)
Kapa library prep kit, by Roche (2 mentions)
Nebnext multiplex oligo, by Illumina (2 mentions)
Tapestation, by Agilent Technologies (2 mentions)
V3 chemistry, by Illumina (1 mentions)
Sureselect target enrichment system, by Agilent Technologies (1 mentions)
Hiseq2500 analyzer, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Nebnext dna library prep reagent set, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Nebnext multiplex oligos, by New England Biolabs (1 mentions)
10 protocols using nimblegenseqcap ez system
1
Genetic Analysis of Congenital Hypogonadotropic Hypogonadism
2
Plasma RNA Extraction and Sequencing
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RNA was extracted from 200 μl plasma using the Agencourt RNAdvance blood kit (Beckman Coulter) eluted into 11 μl of water and then reverse transcribed using Superscript III (Invitrogen) with random hexamers and a NEB Second Strand Synthesis kit (New England BioLabs) for library preparation using the KAPA Library Prep kit (KAPA Biosystems) with index tagging by 16 cycles of PCR using KAPA HiFi HotStart (KAPA Biosystems) and NEBNext Multiplex Oligos (oligonucleotides) for Illumina Index Primer Sets 1 and 2 (New England BioLabs). Libraries were quantified by Qubit (ThermoFisher) and TapeStation (Agilent) and pooled at equimolar concentrations for sequencing on the Illumina MiSeq platform (v3 chemistry).
For capture, pooled G_meta libraries were enriched by either the NimbleGen SeqCap EZ system (Roche) (G_Nimb) or the SureSelect Target Enrichment system (Agilent) (G_SSel), the latter with double-scale reactions and hybridization for 36 h rather than the recommended 16 to 24 h, and then sequenced on the Illumina MiSeq platform using v3 chemistry (Illumina).
For capture, pooled G_meta libraries were enriched by either the NimbleGen SeqCap EZ system (Roche) (G_Nimb) or the SureSelect Target Enrichment system (Agilent) (G_SSel), the latter with double-scale reactions and hybridization for 36 h rather than the recommended 16 to 24 h, and then sequenced on the Illumina MiSeq platform using v3 chemistry (Illumina).
3
Targeted Exome Sequencing of FOXC1 and PITX2
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Genomic DNA was extracted according to the standard phenol–chloroform method. A DNA sample of proband II-1 was used for targeted exome sequencing of the FOXC1 and PITX2 genes. Library construction was performed by the NimbleGen SeqCap EZ System (Roche NimbleGen, Madison, Wisconsin, USA) according to the manufacturer’s instructions. 90 cycle paired-end sequencing was performed on Illumina HiSeq2500 Analyzers (Illumina, San Diego, California, USA) following the manufacturer’s instructions. Illumina Pipeline software (version 1.3.4) was used to perform base-calling and to calculate the quality values for every base.
Reads were aligned to the human reference genome National Center for Biotechnology Information (NCBI) GRCh37 using Burrows-Wheeler Aligner (BWA). Single nucleotide variations (SNVs) and insertions and deletions (indels) identification was performed by SOAPsnp and samtools. SNVs and indels with read depth ≥ 8× and quality ≥30 were reserved for subsequent analysis. Based on the dbSNP database and the 1000 genomes annotation, the polymorphic SNVs were excluded. SNVs and indels affecting coding sequence were annotated using Annotate Variation (ANNOVAR) software.
Reads were aligned to the human reference genome National Center for Biotechnology Information (NCBI) GRCh37 using Burrows-Wheeler Aligner (BWA). Single nucleotide variations (SNVs) and insertions and deletions (indels) identification was performed by SOAPsnp and samtools. SNVs and indels with read depth ≥ 8× and quality ≥30 were reserved for subsequent analysis. Based on the dbSNP database and the 1000 genomes annotation, the polymorphic SNVs were excluded. SNVs and indels affecting coding sequence were annotated using Annotate Variation (ANNOVAR) software.
4
Exome Sequencing of Ear Tissues
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The genomic DNA from ear tissues was fragmented by Covaris technology, sequencing libraries containing fragments of 200-300 bp were constructed, and adapters were ligated to both ends of the fragments for each library. Then, the libraries were subjected to pre-capture PCR, hybridization and capture using the Roche Nimblegen SeqCAP EZ system, which targets ∼60.6 Mb of coding regions. Sequencing was performed on the Illumina HiSeq 2500 platform, generating 126 bp paired-end reads.
5
Exome Sequencing of Genomic DNA
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Genomic DNA was extracted with a standard phenol-chloroform method. Sample libraries were prepared using NEBNext DNA Library Prep Reagent Set for Illumina (New England Biolabs Ltd. catalog# E6000) and subjected to exome capture with NimbleGen SeqCap EZ System (Roche NimbleGen). Paired-end short read sequencing was performed with HiSeq 2000 (Illumina Inc.) at Karolinska Institutet, Sweden.
6
Targeted RNA Sequencing from Plasma
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NGS was carried out using a target enrichment sequencing protocol as previously described.9 Briefly, RNA was extracted from 200 μl plasma using the Agencourt RNAdvance Blood kit (Beckman Coulter) and reverse transcribed using SuperScript III (Invitrogen) with random hexamers and a NEB Second Strand Synthesis kit (New England Biolabs). Adapter-ligated DNA was amplified in real-time on an ABI 7500 cycler, using a KAPA Hifi Real-time library amplification kit. Index tags were added using NEBnext multiplex oligos (New England BioLabs). Pooled libraries were enriched using the NimbleGen SeqCap EZ system (Roche). Amplified DNA was purified using AMPure XP beads and eluted in a final volume of 15 μl. AnAgilent 2200 TapeStation was used to verify the final size profile of amplified library DNA. DNA libraries with appropriate index tags were pooled and paired end sequencing carried out on an Illumina MiSeq instrument using 300-cycle v2 reagents. De novo assembly was carried out using dipSPAdes and mapping with Tanoti (http://www.bioinformatics.cvr.ac.uk/tanoti.php ). Sequence data were submitted to GenBank (accession numbers to follow).
7
Plasma RNA Extraction and Sequencing
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RNA was extracted from 200 µl plasma using the Agencourt RNAdvance blood kit (Beckman Coulter), eluted into 11 μl of water and then reverse transcribed using Superscript III (Invitrogen). Random hexamers and a NEB Second Strand Synthesis kit (New England BioLabs) were used to generate double-stranded DNA. For subsequent library preparation, we used the KAPA Library Prep kit (KAPA Biosystems) with index tagging. DNA was amplified for 16 cycles by polymerase chain reaction using KAPA HiFi HotStart (KAPA Biosystems) and NEBNext Multiplex Oligos (oligonucleotides) for Illumina Index Primer Sets 1 and 2 (New England BioLabs). Libraries were quantified by Qubit (ThermoFisher), sized by TapeStation (Agilent), and pooled at equimolar concentrations. For capture, pooled libraries were enriched with the NimbleGen SeqCap EZ system (Roche) and then sequenced on an Illumina platform.
8
Targeted Exome Sequencing for DSD
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Genomic DNA was extracted from peripheral blood leukocytes using the Qiagen DNA Blood kit (Qiagen, Dusseldorf, Germany). The gene panel (NimblegenSeqCap EZ system, Roche, Basel, Switzerland) was designed to capture all exons and 50 bp flanking intron sequences of the 83 DSD-related genes. The DNA samples were analyzed using massive parallel sequencing (100-bp paired-end reads) on an Illumina HiSeq2500 sequencing system (Illumina, Inc., San Diego, CA, USA) after hybridization to the capture array. Bioinformatic analysis including quality control, read alignment, and variant calling (including single-nucleotide variants [SNVs] and small indels) were performed using bioinformatic pipelines previously described.17 (link) The variants identified by NGS were validated using Sanger sequencing.
9
Genetic Variant Identification in DSD
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Targeted next-generation sequencing and Sanger sequencing were used to identify and validate the gene mutation, respectively. The gene panel (NimblegenSeqCap EZ system, Roche, Basel, Switzerland) was designed to capture all exons and 50-bp flanking intron sequences of 83 DSD-related genes [24 (link)]. A variant was recognized as an underlying disease-causing variant when it was not found in dbSNP, ExAC, GnomAD, and Ensemble database and in 500 Chinese controls, or alternatively, when the allele frequency was found to be less than 0.001 in the database. The amino acid sequence alignment of human NR5A1 with that of other 14 species was performed using UniProt.
10
Targeted exome sequencing for cataract
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DNA of proband III:1 was used for targeted exome sequencing of 139 cataract-related genes. Library construction was performed by the customed Roche NimbleGen SeqCap EZ System (Roche, Madison, Wisconsin, USA), and 90-cycles Paired-end sequencing was conducted on Illumina HiSeq2500 Analyzers (Illumin, San Diego, California, USA). Read mapping and variant analysis procedures were as described previously (Li et al. 2016b ). On the basis of dbSNP database (www.ncbi.nlm.nih.gov/SNP/), Exome Variant Server database (http://evs.gs.washington.edu/EVS/), the Human Gene Mutation Database (http://www.hgmd.cf.ac.uk/ac/index.php) and 1,000 Genomes (http://browser.1000genomes.org/index.html) annotation, the high-frequency (minor allele frequency ≥ 0.01) polymorphism variations were excluded. To perform functional predictions, we used the software Scale-invariant feature transform (SIFT), Polymorphism Phenotyping v2 (PolyPhen 2), and MutationTaster to score these variants. For conservative prediction Clustal Omega program (https://www.ebi.ac.uk/Tools/msa/clustalo/) was made.
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