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I recently did some sequencing on a MiniSeq and unexpectedly the Undetermined_S0_L001_R1_001.fastq.gz file contained quite a lot of data (about 40% of total). I would like to know which indices are present in this file and ideally get a frequency of all index sequences in this file. How can I do this?

This is the output of head:

@MN00148:128:000H2YCW2:1:11102:12116:1065 1:N:0:0
GNAGAGGATTCAGGGAGCCAGCGCGCATATATCAG
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F#FFFFFFFFFFFFFFFFFFA=/A///////////
@MN00148:128:000H2YCW2:1:11102:11354:1065 1:N:0:0
GNACCCGTGCGGCTGGAGAGACCACTAGAAAGAAT
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A#AFFFFFFFFFFFFFFFFF///////FF=////6
@MN00148:128:000H2YCW2:1:11102:4103:1066 1:N:0:0
ANGTTGGTGGTACATCAGGGACGCGGAGATCTCAG
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    $\begingroup$ I do not recall how the formatting of the header exactly is, but the index read sequences should be in there and it is rather trivial to parse and then uniq -c them. $\endgroup$
    – user3051
    May 17, 2021 at 9:36
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    $\begingroup$ How did you demultiplex? With bcl2fastq? You can generate the index reads like I1 $\endgroup$
    – Chris_Rands
    May 17, 2021 at 17:07

2 Answers 2

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I don't know that you can get them from that file. I've noticed that when the Miseq is left to run its version of fastq generation software, there are no indices in the undetermined file. I usually run bcl2fastq myself if I want that information.

They recently came out with a new version of software for the MiSeq, so things might be different now.

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I actually went back to the output folder on the MiniSeq and found this file: DemultiplexSummaryF1L1, which contains demultiplexing information of the whole run. Half-way through this file I found what I was looking for:

### Most Popular Index Sequences                        

### Columns: Sequence ReverseComplement HitCount                        

Index                       
TGACCA  TGGTCA  2455964             
CGATGT  ACATCG  2320579             
GGCCCC  GGGGCC  2023652             
CGCGGG  CCCGCG  1983605             
CCGTCC  GGACGG  1641274         
CAGATC  GATCTG  1514252             
CCGGCC  GGCCGG  1319383             
CCGCGC  GCGCGG  1109940             
ACAGTG  CACTGT  891344      
CCCGGG  CCCGGG  706083              
TGCCCA  TGGGCA  168294              
CAGCTC  GAGCTG  164091  
.... [data continues]
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