Take Home Message
• The different sequence related formats include different information about the
sequence.
• SRA is the file format in which all NCBI SRA content is provided.
• NGS sequence text files should be stored compressed to save up hard drive space.
• Quality Check of your raw reads is an essential step before further analysis.
• If any adapter contamination or low-quality bases at the end of your obtained
sequencing reads are detected by the Quality Check tool, they should be removed.
Further Reading
• Sequence Read Archive Submissions Staff. Using the SRA Toolkit to convert *.sra files
into other formats. In: SRA Knowledge Base [Internet]. Bethesda (MD): National
Center for Biotechnology Information (US); 2011-. Available from: https://www.ncbi.
nlm.nih.gov/books/NBK158900/
Answers to Review Questions:
Answer to Question 1: whole genome: [# of sequenced bases] / [size of genome]
300M x 2 x 100
ð
Þ
3:2 GB
¼
45 x 10
9
3:2 x 10
9 ¼ 14.0625 (coverage)
Answer to Question 2: Homo sapiens, transcriptome, Illumina HiSeq2500, 51,
paired-end.
Answer to Question 3: 2 19 28 35 66 35 35 35 39 37 39 37 37 39 40 38 39 40 40 40
Answer to Question 4: To create a reference index to be able to perform sequence
alignment.
Acknowledgements We thank Patricia Basurto and Carolina Castañeda of the International Laboratory for Human Genome Research (National Autonomous University of Mexico, Juriquilla campus) for reviewing this chapter.
References
1. Sims D, Sudbery I, Ilott NE, Heger A, Ponting CP. Sequencing depth and coverage: key
considerations in genomic analyses. Nat Rev Genetics. 2014;15(2):121–32.
2. Cock PJ, Fields CJ, Goto N, Heuer ML, Rice PM. The Sanger FASTQ file format for sequences
with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res. 2010;38
(6):1767–71.
3. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map
format and SAMtools. Bioinformatics. 2009;25(16):2078–9.
7 NGS Data
103
• The different sequence related formats include different information about the
sequence.
• SRA is the file format in which all NCBI SRA content is provided.
• NGS sequence text files should be stored compressed to save up hard drive space.
• Quality Check of your raw reads is an essential step before further analysis.
• If any adapter contamination or low-quality bases at the end of your obtained
sequencing reads are detected by the Quality Check tool, they should be removed.
Further Reading
• Sequence Read Archive Submissions Staff. Using the SRA Toolkit to convert *.sra files
into other formats. In: SRA Knowledge Base [Internet]. Bethesda (MD): National
Center for Biotechnology Information (US); 2011-. Available from: https://www.ncbi.
nlm.nih.gov/books/NBK158900/
Answers to Review Questions:
Answer to Question 1: whole genome: [# of sequenced bases] / [size of genome]
300M x 2 x 100
ð
Þ
3:2 GB
¼
45 x 10
9
3:2 x 10
9 ¼ 14.0625 (coverage)
Answer to Question 2: Homo sapiens, transcriptome, Illumina HiSeq2500, 51,
paired-end.
Answer to Question 3: 2 19 28 35 66 35 35 35 39 37 39 37 37 39 40 38 39 40 40 40
Answer to Question 4: To create a reference index to be able to perform sequence
alignment.
Acknowledgements We thank Patricia Basurto and Carolina Castañeda of the International Laboratory for Human Genome Research (National Autonomous University of Mexico, Juriquilla campus) for reviewing this chapter.
References
1. Sims D, Sudbery I, Ilott NE, Heger A, Ponting CP. Sequencing depth and coverage: key
considerations in genomic analyses. Nat Rev Genetics. 2014;15(2):121–32.
2. Cock PJ, Fields CJ, Goto N, Heuer ML, Rice PM. The Sanger FASTQ file format for sequences
with quality scores, and the Solexa/Illumina FASTQ variants. Nucleic Acids Res. 2010;38
(6):1767–71.
3. Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, et al. The sequence alignment/map
format and SAMtools. Bioinformatics. 2009;25(16):2078–9.
7 NGS Data
103