Kun:LabNotes/SingleCellExpr/2013-1-1

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HL140.2 run data analysis[edit]

Lane 2[edit]

  • The sequencing libraries were prepared by Rui on Ambion Human Brain Reference (HBR) RNA diluted to various levels [1].
  • The multiplexed library was sequenced in Lane 2 of the HL140 flowcell. The first run of this flowcell failed due to low-quality reads towards the second half of Read 1. This flowcell was repeated in 121226_HL140.2.
  • Data analysis is performed on 80+8+8bp reads.

Demultiplexing[edit]

  • Rui used a combination of three different barcodes for this batch of libraries. Will first decode using the standard N2 index, then with the 8bp barcodes in the T20 primers (correspond to bases 1-8 in Read 2).
  • Run the following command in /home/kunzhang/seqStore/121226_HL140.2: 38.1 million of 40.2 million quality-filtered reads were decoded.
   qseq2fastqSplitIndxPE96plexGAIIx.pl  /media/FreshReads/121226_HL140_2/Data/Intensities/Bustard1.9.4_01-01-2013_root s_2 lane2_decoded > s2_demultiplexing.log &
  • In the /home/kunzhang/RNAseq/Data/121226_HL140.2/RL_HBR directory, create eight folders for the eight indexes (N2_indx01, N2_indx02, ..., N2_indx08). Then run the following commands:
 ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx1.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx1.txt N2_indx01 > N2_indx01_t20bc8.log &
 ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx2.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx2.txt N2_indx02 > N2_indx02_t20bc8.log &
 ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx3.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx3.txt N2_indx03 > N2_indx03_t20bc8.log &
 ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx6.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx6.txt N2_indx06 > N2_indx06_t20bc8.log &
 ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx4.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx4.txt N2_indx04 > N2_indx04_t20bc8.log &
 ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx5.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx5.txt N2_indx05 > N2_indx05_t20bc8.log &
 ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx7.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx7.txt N2_indx07 > N2_indx07_t20bc8.log &
 ./fastq_decon_T20BC8.pl /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_1_Indx8.txt /home/kunzhang/seqStore/121226_HL140.2/lane2_decoded/s_2_2_Indx8.txt N2_indx08 > N2_indx08_t20bc8.log &
  The 85%-90% of the reads were successfully decoded.
  N2_index    # total reads  # decoded reads  % decoded
  N2_indx01    4191926       3752364          89.5%
  N2_indx02    3539471       3161288          89.3%
  N2_indx03    5579440       4951974          88.8%
  N2_indx04    5649610       5053323          89.4%
  N2_indx05    4926765       4314315          87.6%
  N2_indx06    2879929       2416443          83.9%
  N2_indx07    7703629       6637497          86.2%
  N2_indx08    3696754       3148274          85.2%