Multiplexing Index 1 (i7) is always read. Index 2 (i5) is read only in dual index setting. Single indexing Dual indexing Single end (SE) R1, R2 R1, R2, R3 Paired end (PE) R1, R2, R4 R1, R2, R3, R4 Multiplexing - dual index reads - paired end
To prepare genome reference in FASTA format for mouse assembly NCBI37/mm9, we have two options: From UCSC Using the mm9 assembly from UCSC golden Path. Do not use the masked file chromFaMasked.tar.gz! 1# download `chromFa.tar.gz ` from UCSC golden path 2wget http://hgdownload.cse.ucsc.edu/goldenPath/mm9/bigZips/chromFa.tar.gz 3#or 4sync -avzP rsync://hgdownload.cse.ucsc.edu/goldenPath/mm9/bigZips/chromFa.tar.gz . 5 6# uncompress the downloaded file 7tar -xvzf chromFa.tar.gz 8 9# remove `*random.fa` chromosomes 10rm -rf *_random.fa 11 12# concatenate all FASTA files into a single file 13cat *.fa > mm9.fa 14 15# index the concatenated .fa file using `samtools` 16module load samtools # required at HPC 17samtools faidx mm9.fa From Ensembl Using Mus Musculus release-67 (Ensembl) 1# download the following folder from Ensembl 2lftp ftp://ftp.ensembl.org/pub/release-67/fasta/mus_musculus/dna/ 3mget * 4 5# uncompress the downloaded *.fa.gz files 6gzip -d *.fa.gz 7 8# delete the masked version of the genome sequence which contains '_rm' in the name 9rm -rf *_rm* 10 11# concatenate all FASTA files into a single file 12cat *.fa > mm9Ensembl.fa 13 14# index the concatenated .fa file using `samtools` 15module load samtools # required at HPC 16samtools faidx mm9Ensembl.fa One of the main differences between the two sources, which is very important for downstream applications, is chromosome annotation. Here is a comparison between the header lines, also known as the identifier or description lines, used in the FASTA files from both sources. While UCSC uses the chr prefix in front of the chromosome number, Ensembl merely uses the chromosome number. 1# mm9 from UCSC 2~/f/G/N/mm9_fasta ❯❯❯ cat mm9.fa | grep '>' 3>chr10 4>chr11 5>chr12 6>chr13 7>chr14 8>chr15 9>chr16 10>chr17 11>chr18 12>chr19 13>chr1 14>chr2 15>chr3 16>chr4 17>chr5 18>chr6 19>chr7 20>chr8 21>chr9 22>chrM 23>chrX 24>chrY 25 26# ENS67 from Ensembl 27~/f/G/N/ENS67_fasta ❯❯❯ cat ENS67.fa | grep '>' 28>10 dna:chromosome chromosome:NCBIM37:10:1:129993255:1 29>11 dna:chromosome chromosome:NCBIM37:11:1:121843856:1 30>12 dna:chromosome chromosome:NCBIM37:12:1:121257530:1 31>13 dna:chromosome chromosome:NCBIM37:13:1:120284312:1 32>14 dna:chromosome chromosome:NCBIM37:14:1:125194864:1 33>15 dna:chromosome chromosome:NCBIM37:15:1:103494974:1 34>16 dna:chromosome chromosome:NCBIM37:16:1:98319150:1 35>17 dna:chromosome chromosome:NCBIM37:17:1:95272651:1 36>18 dna:chromosome chromosome:NCBIM37:18:1:90772031:1 37>19 dna:chromosome chromosome:NCBIM37:19:1:61342430:1 38>1 dna:chromosome chromosome:NCBIM37:1:1:197195432:1 39>2 dna:chromosome chromosome:NCBIM37:2:1:181748087:1 40>3 dna:chromosome chromosome:NCBIM37:3:1:159599783:1 41>4 dna:chromosome chromosome:NCBIM37:4:1:155630120:1 42>5 dna:chromosome chromosome:NCBIM37:5:1:152537259:1 43>6 dna:chromosome chromosome:NCBIM37:6:1:149517037:1 44>7 dna:chromosome chromosome:NCBIM37:7:1:152524553:1 45>8 dna:chromosome chromosome:NCBIM37:8:1:131738871:1 46>9 dna:chromosome chromosome:NCBIM37:9:1:124076172:1 47>MT dna:chromosome chromosome:NCBIM37:MT:1:16299:1 48>X dna:chromosome chromosome:NCBIM37:X:1:166650296:1 49>Y dna:chromosome chromosome:NCBIM37:Y:1:15902555:1