The diploid genome sequence of a Chinese individual was sequenced


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Accession number: ERA000005

Eight single-end and two paired-end libraries.

Illumina Genome Analysers 

The read lengths averaged 35 base pairs 

2 paired-end libraries had a span size of 135 bp and 440 bp

3.3 billion reads 

117.7 gigabases (Gb) of sequence. 72 Gb from single-end reads and 45.7 Gb from paired-end reads.

Program used: Short Oligonucleotide Alignment Program (SOAP)

102.9 Gb of sequence (87.4% of all data) was properly aligned to the NCBI human reference genome (build 36.1; hereafter called NCBI36). 

36-fold average coverage of NCBI36

The effective genome coverage of the single- and paired-end sequencing was 22.5-fold and 13.5-fold, respectively.

99.97% of NCBI36 (excluding Ns, which are undetermined sequence of the reference genome) was covered by at least one uniquely or repeatedly aligned read (uniquely aligned reads had only one best hit on NBCI36; repeatedly aligned reads had multiple possible alignments; see Methods for details).

Average per-nucleotide difference of 1.45% from the NCBI36 sequence

YH genome consensus sequence covered 92% of the NCBI36 sequence (92.6% of the autosomes; 83.1% of the sex chromosomes), 

3.07 million SNPs. 

The remaining 8% of the reference sequence was composed of either repetitive sequence (6.6%) that did not have any uniquely mapped reads or sequence that didn't pass our filtering steps (1.4%).

2.26 million (73.5%) of the YH SNPs were present in dbSNP as validated SNPs, and 0.4 million (12.9%) were present as non-validated SNPs.
0.42 million SNPs were novel 

They also did Illumina 1M BeadChip for genotyping

They used polymerase chain reaction (PCR) amplification and traditional Sanger sequencing technology on a subset of the inconsistent SNPs and small indels to determine whether they conformed to the genotyping or GA sequencing results 

The use of paired-end reads as opposed to single-end reads further reduces SNP calling errors. Of note, SNP calling errors of homozygous and heterozygous SNPs differ significantly.

Unique to the 3 genomes sequenced: for YH, 978,370 (31.8%) SNPs; for Venter, 924,333 (30.1%); and for Watson, 1,096,873 (33.0%)

For Indels

For indel identification, they required at least 3 pairs of reads to define an indel. They only considered paired-end read-gapped alignments that had insertion or deletion sizes of 3 bp or less to avoid creating alignment errors. Confining indel size was necessary to obtain the best detection accuracy given our short-read sequencing strategy. From this analysis, they identified a total of 135,262 indels.

Most (59.1%) of the indels were novel 

Depth effect on genome sequencing
To determine what sequencing depth provides the best genome coverage and lowest SNP-calling error rates for a diploid human genome, they randomly extracted subsets of reads with different average depths from all the mapped reads on chromosome 12, which has a relatively moderate number of repeats. 
SNPs were identified using GA sequencing and then compared with the genotyping data. 
They applied the same filtering steps as used in SNP identification.

At a depth greater than 10-fold, the assembled consensus covered 83.63% of the NCBI reference genome using single-end reads and 95.88% coverage using paired-end reads. Thus, greater sequencing depth provides only a small increase in genome coverage.

Evolution and selection
Alleles that are identical between two random individuals are more likely to be the most common type of allele in the population

The YH individual was estimated to share alleles (thus ancestry) at 94.12% with the Asian, 4.12% with the European and 1.76% with the African populations

effective Chinese population size is about 5,700

 3,300 for the effective human population size


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